CesA Proteins Research Articles

Discovery and Characterization of Fluopipamine, a Putative Cellulose Synthase 1 Antagonist within Arabidopsis.

Herbicide-resistant weeds are increasingly a problem in crop fields when exposed to similar chemistry over time. To avoid future yield losses, identifying herbicidal chemistry needs to be accelerated. We screened 50,000 small molecules using a liquid-handling robot and light microscopy focusing on pre-emergent herbicides in the family of cellulose biosynthesis inhibitors. Through phenotypic, chemical, genetic, and in silico methods we uncovered 6-{[4-(2-fluorophenyl)-1-piperazinyl]methyl}-N-(2-methoxy-5-methylphenyl)-1,3,5-triazine-2,4-diamine (fluopipamine). Symptomologies support fluopipamine as a putative antagonist of cellulose synthase enzyme 1 (CESA1) from Arabidopsis (Arabidopsis thaliana). Ectopic lignification, inhibition of etiolation, phenotypes including loss of anisotropic cellular expansion, swollen roots, and live cell imaging link fluopipamine to cellulose biosynthesis inhibition. Radiolabeled glucose incorporation of cellulose decreased in short-duration experiments when seedlings were incubated in fluopipamine. To elucidate the mechanism, ethylmethanesulfonate mutagenized M2 seedlings were screened for fluopipamine resistance. Two loci of genetic resistance were linked to CESA1. In silico docking of fluopipamine, quinoxyphen, and flupoxam against various CESA1 mutations suggests that an alternative binding site at the interface between CESA proteins is necessary to preserve cellulose polymerization in compound presence. These data uncovered potential fundamental mechanisms of cellulose biosynthesis in plants along with feasible leads for herbicidal uses.

Genome-Wide Identification and Expression Analysis of CesA Gene Family in Corymbia citriodora

The CesA proteins are key players in cellulose synthesis. Eucalyptus is well-known for cellulose synthesis, although the mechanism of cellulose synthesis is largely unknown. In this study, 11 CcCesA genes were identified by comprehensive bioinformatics analysis in Corymbia citriodora, a eucalypt species. CcCesA genes were generally conserved in each subfamily. Among them, four genes lacked a transmembrane domain in the N-terminal. We showed that genes of CesA2, 4, 5, and 8 may regulate the synthesis of secondary cell walls (SCWs) through phylogenetic trees analysis. Two pairs of segmental duplication and one pair of tandem duplication were detected in CcCesAs. Analysis of Ka/Ks ratios revealed that the duplicated genes were under negative or purifying selection. Numerous cis-acting elements related to plant hormones and light reactions were identified in the promoters of CcCesAs. Expression analysis confirmed that genes of CesA2, 4, and 8 regulate the synthesis of SCWs, and CesA8, in particular, had the highest expression compared with other genes. The results will help us understand the complexity of the CcCesAs in different diameters at breast height and provide valuable information for future functional characterization of specific genes in C. citriodora.

Transcriptome Mining Provides Insights into Cell Wall Metabolism and Fiber Lignification in Agave tequilana Weber.

Resilience of growing in arid and semiarid regions and a high capacity of accumulating sugar-rich biomass with low lignin percentages have placed Agave species as an emerging bioenergy crop. Although transcriptome sequencing of fiber-producing agave species has been explored, molecular bases that control wall cell biogenesis and metabolism in agave species are still poorly understood. Here, through RNAseq data mining, we reconstructed the cellulose biosynthesis pathway and the phenylpropanoid route producing lignin monomers in A. tequilana, and evaluated their expression patterns in silico and experimentally. Most of the orthologs retrieved showed differential expression levels when they were analyzed in different tissues with contrasting cellulose and lignin accumulation. Phylogenetic and structural motif analyses of putative CESA and CAD proteins allowed to identify those potentially involved with secondary cell wall formation. RT-qPCR assays revealed enhanced expression levels of AtqCAD5 and AtqCESA7 in parenchyma cells associated with extraxylary fibers, suggesting a mechanism of formation of sclerenchyma fibers in Agave similar to that reported for xylem cells in model eudicots. Overall, our results provide a framework for understanding molecular bases underlying cell wall biogenesis in Agave species studying mechanisms involving in leaf fiber development in monocots.

Expression Patterns and Gene Analysis of the Cellulose Synthase Gene Superfamily in Eucalyptus grandis

Cellulose is the world’s most abundant renewable energy resource, and a variety of cellulose synthase genes are involved in the biosynthesis of cellulose. In the process of cellulose synthesis, all cellulose synthases are interrelated and act synergistically. In this study, we analyzed the contents of cellulose, hemicellulose, and lignin in the different parts and tissues of E. grandis. The results showed that the cellulose content had greater differences among three different heights. On this basis, we carried out the transcriptome-wide profiling of gene expression patterns using RNA sequencing. A total of 2066 differentially expressed genes were identified for three pairwise comparisons between three different heights, most of which were related to the programmed photosynthetic membrane and photosystem. A total of 100 transcripts of CSs (58 CesA and 42 Csl) were obtained from transcriptome libraries. The expression pattern of these genes indicated that different CS genes had a wide range of expression profiles. A phylogenetic analysis of 135 reference CS genes showed that the CSs of E. grandis were clustered into six major groups (CesA1-9, CslA, CslB/H, CslD, CslE, and CslG). Based on the weighted gene co-expression network analysis, a dual-directional regulation mechanism between Csl and CesA proteins in the cellulose biosynthesis was identified. The gene expression profile analysis, using qRT-PCR in different tissues of E. grandis, demonstrated that the CSs were highly expressed in xylem, and CesAs had a higher relative expression than Csls. The analysis of sequence similarity combined with the expression pattern indicated that the CesA1, 3, and 6 transcripts were associated with the biosynthesis of the secondary cell wall, and CesA4, 5, and 7 transcripts were more likely to associate with the biosynthesis of the primary cell wall. Finally, the qRT-PCR analysis confirmed the expression of 11 selected CSs in three different parts of E. grandis.

Analysis of a cellulose synthase catalytic subunit from the oomycete pathogen of crops Phytophthora capsici

Phytophthora capsici Leonian is an important oomycete pathogen of crop vegetables, causing significant economic losses each year. Its cell wall, rich in cellulose, is vital for cellular integrity and for interactions with the host organisms. Predicted cellulose synthase (CesA) proteins are expected to catalyze the polymerization of cellulose, but this has not been biochemically demonstrated in an oomycete. Here, we present the properties of the four newly identified CesA proteins from P. capsici and compare their domain organization with that of CesAs from other lineages. Using a newly constructed glucosyltransferase-deficient variant of Saccharomyces cerevisiae with low residual background activity, we have achieved successful heterologous expression and biochemical characterization of a CesA protein from P. capsici (PcCesA1). Our results demonstrate that the individual PcCesA1 enzyme produces cellobiose as the major reaction product. Co-immunoprecipitation studies and activity assays revealed that several PcCesA proteins interact together to form a complex whose multiproteic nature is most likely required for cellulose microfibril formation. In addition to providing important insights into cellulose synthesis in the oomycetes, our data may assist the longer term identification of cell wall biosynthesis inhibitors to control infection by pathogenic oomycetes.

Heterologous overexpression, purification and functional analysis of plant cellulose synthase from green bamboo

BackgroundThe cellulose synthase complex (CSC), composed of cellulose synthase (CesA) proteins, is a catalytic enzyme complex involved in cellulose synthesis in the plant cell. CesA proteins synthesize cellulose microfibrils corresponding to the microtubule direction and export linear products across the plasma membrane. However, the CSC arrangement and the mechanism of cellulose synthesis in plant cells remain unclear. Purified CesA proteins are required to determine biochemical and biophysical characteristics.ResultsIn this study, we constructed, expressed, and purified six heterologously expressed cellulose synthases from Bambusa oldhamii (BoCesA) and analyzed the associated enzyme activity. The conjugating sequences of the maltose-binding protein (MBP) gene and the BoCesA genes were constructed into the expression vector pYES2/CT and were further transformed into yeast cells (BCY123) for fermentation culturing. Purified BoCesA recombinant proteins were obtained by a two-step purification procedure, consisting of immobilized metal affinity chromatography to purify MBP-BoCesAs and size-exclusion chromatography (Superdex-200) to isolate BoCesAs in oligomeric form. The enzymatic activity of oligomeric BoCesAs with 80% purity was determined by partially methylated alditol acetate (PMAA)-coupled gas chromatography–mass spectrometry (GC–MS) analysis. Furthermore, the long fiber-like products synthesized by oligomeric BoCesAs were observed under a transmission electron microscope (TEM) and were further confirmed as cellulose microfibril products.ConclusionsIn this study, we successfully established a heterologous expression and purification system for BoCesAs. The purified recombinant BoCesA proteins display enzyme activity and can produce protein in milligram quantities for further studies on molecular composition and structure.

Exploiting CELLULOSE SYNTHASE (CESA) Class Specificity to Probe Cellulose Microfibril Biosynthesis.

Cellulose microfibrils are the basic units of cellulose in plants. The structure of these microfibrils is at least partly determined by the structure of the cellulose synthase complex. In higher plants, this complex is composed of 18 to 24 catalytic subunits known as CELLULOSE SYNTHASE A (CESA) proteins. Three different classes of CESA proteins are required for cellulose synthesis and for secondary cell wall cellulose biosynthesis these classes are represented by CESA4, CESA7, and CESA8. To probe the relationship between CESA proteins and microfibril structure, we created mutant cesa proteins that lack catalytic activity but retain sufficient structural integrity to allow assembly of the cellulose synthase complex. Using a series of Arabidopsis (Arabidopsis thaliana) mutants and genetic backgrounds, we found consistent differences in the ability of these mutant cesa proteins to complement the cellulose-deficient phenotype of the cesa null mutants. The best complementation was observed with catalytically inactive cesa4, while the equivalent mutation in cesa8 exhibited significantly lower levels of complementation. Using a variety of biophysical techniques, including solid-state nuclear magnetic resonance and Fourier transform infrared microscopy, to study these mutant plants, we found evidence for changes in cellulose microfibril structure, but these changes largely correlated with cellulose content and reflected differences in the relative proportions of primary and secondary cell walls. Our results suggest that individual CESA classes have similar roles in determining cellulose microfibril structure, and it is likely that the different effects of mutating members of different CESA classes are the consequence of their different catalytic activity and their influence on the overall rate of cellulose synthesis.

Synthesis and Self-Assembly of Cellulose Microfibrils from Reconstituted Cellulose Synthase.

Cellulose, the major component of plant cell walls, can be converted to bioethanol and is thus highly studied. In plants, cellulose is produced by cellulose synthase, a processive family-2 glycosyltransferase. In plant cell walls, individual β-1,4-glucan chains polymerized by CesA are assembled into microfibrils that are frequently bundled into macrofibrils. An in vitro system in which cellulose is synthesized and assembled into fibrils would facilitate detailed study of this process. Here, we report the heterologous expression and partial purification of His-tagged CesA5 from Physcomitrella patens Immunoblot analysis and mass spectrometry confirmed enrichment of PpCesA5. The recombinant protein was functional when reconstituted into liposomes made from yeast total lipid extract. The functional studies included incorporation of radiolabeled Glc, linkage analysis, and imaging of cellulose microfibril formation using transmission electron microscopy. Several microfibrils were observed either inside or on the outer surface of proteoliposomes, and strikingly, several thinner fibrils formed ordered bundles that either covered the surfaces of proteoliposomes or were spawned from liposome surfaces. We also report this arrangement of fibrils made by proteoliposomes bearing CesA8 from hybrid aspen. These observations describe minimal systems of membrane-reconstituted CesAs that polymerize β-1,4-glucan chains that coalesce to form microfibrils and higher-ordered macrofibrils. How these micro- and macrofibrils relate to those found in primary and secondary plant cell walls is uncertain, but their presence enables further study of the mechanisms that govern the formation and assembly of fibrillar cellulosic structures and cell wall composites during or after the polymerization process controlled by CesA proteins.

Genome-wide identification, subcellular localization and gene expression analysis of the members of CESA gene family in common tobacco (Nicotiana tabacum L.).

Cellulose-synthase proteins (CESAs) are membrane localized proteins and they form protein complexes to produce cellulose in the plasma membrane. CESA proteins play very important roles in cell wall construction during plant growth and development. In this study, a total of 21 NtCESA gene sequences were identified by using PF03552 conserved protein sequence and 10 AtCESA protein sequences of Arabidopsis thaliana to blast against the common tobacco (Nicotiana tabacum L.) genome database with TBLASTN protocol. We analyzed the physical and chemical properties of protein sequences based on some software or on-line analysis tools. The results showed that there were no significant variances in terms of the physical and chemical properties of the 21 NtCESA proteins. First, phylogenetic tree analysis showed that 21 NtCESA genes and 10 AtCESA genes were clustered into five groups, and the gene structures were similar among the genes that are clustered into the same group. Second, in all of the 21 NtCESA proteins the conserved zinc finger domain was identified in the N-terminus, transmembrane domains were identified in the C-terminus and the DDD-QXXRW conserved domains were also identified. Third, gene expression analysis results indicated that most NtCESA genes were expressed in roots and leaves of seedling or mature tissues of tobacco, seeds and callus tissues. The genes that clustered into the same group share similar expression patterns. Importantly, NtCESA proteins that are involved in secondary cell wall cellulose synthesis have two extra transmembrane domains compared with that involved in primary cell wall cellulose biosynthesis. In addition, subcellular localization results showed that NtCESA9 and NtCESA14 were two plasma membrane anchored proteins. This study will lay a foundation for further functional characterization of these NtCESA genes.

Functional Analysis of Cellulose Synthase (CESA) Protein Class Specificity.

The cellulose synthase complex (CSC) exhibits a 6-fold symmetry and is known as a "rosette." Each CSC is believed to contain between 18 and 24 CESA proteins that each synthesize an individual glucan chain. These chains form the microfibrils that confer the remarkable structural properties of cellulose. At least three different classes of CESA proteins are essential to form the CSC However, while organization of the CSC determines microfibril structure, how individual CESA proteins are organized within the CSC remains unclear. Parts of the plant CESA proteins map sufficiently well onto the bacterial CESA (BcsA) structure, indicating that they are likely to share a common catalytic mechanism. However, plant CESA proteins are much larger than the bacterial BcsA protein, prompting the suggestion that these plant-specific regions are important for interactions between CESA proteins and for conferring CESA class specificity. In this study, we have undertaken a comprehensive analysis of well-defined regions of secondary cell wall CESA proteins, with the aim of defining what distinguishes different CESA proteins and hence what determines the specificity of each CESA class. Our results demonstrate that CESA class specificity extends throughout the protein and not just in the highly variable regions. Furthermore, we find that different CESA isoforms vary greatly in their levels of site specificity and this is likely to be determined by the constraints imposed by their position within the CSC rather than their primary structure.

Novel Structural and Functional Motifs in cellulose synthase (CesA) Genes of Bread Wheat (Triticum aestivum, L.).

Cellulose is the primary determinant of mechanical strength in plant tissues. Late-season lodging is inversely related to the amount of cellulose in a unit length of the stem. Wheat is the most widely grown of all the crops globally, yet information on its CesA gene family is limited. We have identified 22 CesA genes from bread wheat, which include homoeologs from each of the three genomes, and named them as TaCesAXA, TaCesAXB or TaCesAXD, where X denotes the gene number and the last suffix stands for the respective genome. Sequence analyses of the CESA proteins from wheat and their orthologs from barley, maize, rice, and several dicot species (Arabidopsis, beet, cotton, poplar, potato, rose gum and soybean) revealed motifs unique to monocots (Poales) or dicots. Novel structural motifs CQIC and SVICEXWFA were identified, which distinguished the CESAs involved in the formation of primary and secondary cell wall (PCW and SCW) in all the species. We also identified several new motifs specific to monocots or dicots. The conserved motifs identified in this study possibly play functional roles specific to PCW or SCW formation. The new insights from this study advance our knowledge about the structure, function and evolution of the CesA family in plants in general and wheat in particular. This information will be useful in improving culm strength to reduce lodging or alter wall composition to improve biofuel production.

Protocol: a medium-throughput method for determination of cellulose content from single stem pieces of Arabidopsis thaliana.

BackgroundLignocellulosic biomass is an important renewable resource for biofuels and materials. How plants synthesise cellulose is not completely understood. It is known that cellulose synthase complex (CSCs) moving in the plasma membrane synthesise the cellulose. CESA proteins are the core components of CSC. In Arabidopsis, in vitro mutagenesis of proteins followed by complementation analysis of mutants lacking the gene represents an important tool for studying any biological process, including cellulose biosynthesis. Analysis of a large number of plants is crucial for these types of studies.ResultsBy using aspiration rather than centrifugation to remove liquids during various stages of protocol, we were able to increase the throughput of the method as well as minimise the sample loss. As a test case, we determined cellulose content of wild type and secondary wall cesa mutants across the length of primary shoot which was fond to be rather uniform in 7-week-old plants. Additionally, we found that the cellulose content of single mutants was comparable to the higher order mutants.ConclusionsHere we describe a medium-throughput adaptation of Updegraff’s method that allowed us to determine cellulose content of 200 samples each week.

In vitro synthesis of cellulose microfibrils by a membrane protein from protoplasts of the non-vascular plant Physcomitrella patens

Plant cellulose synthases (CesAs) form a family of membrane proteins that are associated with hexagonal structures in the plasma membrane called CesA complexes (CSCs). It has been difficult to purify plant CesA proteins for biochemical and structural studies. We describe CesA activity in a membrane protein preparation isolated from protoplasts of Physcomitrella patens overexpressing haemagglutinin (HA)-tagged PpCesA5. Incubating the membrane preparation with UDP-glucose predominantly produced cellulose. Negative-stain EM revealed microfibrils. Cellulase bound to and degraded these microfibrils. Vibrational sum frequency generation (SFG) spectroscopic analysis detected the presence of crystalline cellulose in the microfibrils. Putative CesA proteins were frequently observed attached to the microfibril ends. Combined cross-linking and gradient centrifugation showed bundles of cellulose microfibrils with larger particle aggregates, possibly CSCs. These results suggest that P. patens is a useful model system for biochemical and structural characterization of plant CSCs and their components.

Unidirectional movement of cellulose synthase complexes in Arabidopsis seed coat epidermal cells deposit cellulose involved in mucilage extrusion, adherence, and ray formation.

Cellulose synthase5 (CESA5) synthesizes cellulose necessary for seed mucilage adherence to seed coat epidermal cells of Arabidopsis (Arabidopsis thaliana). The involvement of additional CESA proteins in this process and details concerning the manner in which cellulose is deposited in the mucilage pocket are unknown. Here, we show that both CESA3 and CESA10 are highly expressed in this cell type at the time of mucilage synthesis and localize to the plasma membrane adjacent to the mucilage pocket. The isoxaben resistant1-1 and isoxaben resistant1-2 mutants affecting CESA3 show defects consistent with altered mucilage cellulose biosynthesis. CESA3 can interact with CESA5 in vitro, and green fluorescent protein-tagged CESA5, CESA3, and CESA10 proteins move in a linear, unidirectional fashion around the cytoplasmic column of the cell, parallel with the surface of the seed, in a pattern similar to that of cortical microtubules. Consistent with this movement, cytological evidence suggests that the mucilage is coiled around the columella and unwinds during mucilage extrusion to form a linear ray. Mutations in CESA5 and CESA3 affect the speed of mucilage extrusion and mucilage adherence. These findings imply that cellulose fibrils are synthesized in an ordered helical array around the columella, providing a distinct structure to the mucilage that is important for both mucilage extrusion and adherence.

Powerful regulatory systems and post-transcriptional gene silencing resist increases in cellulose content in cell walls of barley.

BackgroundThe ability to increase cellulose content and improve the stem strength of cereals could have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Here, such potential is explored in the commercially important crop barley through the manipulation of cellulose synthase genes (CesA).ResultsBarley plants transformed with primary cell wall (PCW) and secondary cell wall (SCW) barley cellulose synthase (HvCesA) cDNAs driven by the CaMV 35S promoter, were analysed for growth and morphology, transcript levels, cellulose content, stem strength, tissue morphology and crystalline cellulose distribution. Transcript levels of the PCW HvCesA transgenes were much lower than expected and silencing of both the endogenous CesA genes and introduced transgenes was often observed. These plants showed no aberrant phenotypes. Although attempts to over-express the SCW HvCesA genes also resulted in silencing of the transgenes and endogenous SCW HvCesA genes, aberrant phenotypes were sometimes observed. These included brittle nodes and, with the 35S:HvCesA4 construct, a more severe dwarfing phenotype, where xylem cells were irregular in shape and partially collapsed. Reductions in cellulose content were also observed in the dwarf plants and transmission electron microscopy showed a significant decrease in cell wall thickness. However, there were no increases in overall crystalline cellulose content or stem strength in the CesA over-expression transgenic plants, despite the use of a powerful constitutive promoter.ConclusionsThe results indicate that the cellulose biosynthetic pathway is tightly regulated, that individual CesA proteins may play different roles in the synthase complex, and that the sensitivity to CesA gene manipulation observed here suggests that in planta engineering of cellulose levels is likely to require more sophisticated strategies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0448-y) contains supplementary material, which is available to authorized users.

The Endocytosis of Cellulose Synthase in Arabidopsis Is Dependent on μ2, a Clathrin-Mediated Endocytosis Adaptin

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.

Designing Cell Walls for Improved Bioenergy Production

Dwindling supply and unpredictable price fluctuations of fossil fuels have necessitated a renewed worldwide search for alternative fuels. Plant cell walls, rich in cellulose and hemicellulose, are actively pursued for their use in production of second-generation biofuels. It has been envisioned that degradation of these carbohydrate polymers to their sugar monomer subunits (saccharification) will provide necessary substrates for their yeast-mediated fermentation to ethanol. But the major component of plant cell walls, cellulose, is not easily amenable to this type of deconstruction and is tightly interlinked with hemicelluloses and lignin, making cell walls highly recalcitrant to saccharification.

Importance of two consecutive methionines at the N-terminus of a cellulose synthase (PtdCesA8A) for normal wood cellulose synthesis in aspen

All known orthologs of a secondary wall-associated cellulose synthase (CesA) gene from Arabidopsis, AtCesA8, encode CesA proteins with two consecutive methionines at their N-termini (MM or 2M). Here, we report that these 2Ms in an aspen ortholog of AtCesA8, PtdCesA8A, are important for maintaining normal wood cellulose biosynthesis in aspen trees. Overexpression of an altered PtdCesA8A cDNA encoding a PtdCesA8A protein missing one methionine at the N-terminus (1M) in aspen resulted in substantial decrease in cellulose content and caused negative effects on wood strength, suggesting that both methionines are essential for proper CesA expression and function in developing xylem tissues. Transcripts from a pair of paralogous native PtdCesA8 genes, as well as introduced PtdCesA8A:1M transgenes were significantly reduced in developing xylem tissues of transgenic aspen plants, suggestive of a co-suppression event. Overexpression of a native PtdCesA8A cDNA encoding a CesA protein with 2Ms at the N-terminus did not cause any such phenotypic changes. These results suggest the importance of 2Ms present at the N-terminus of PtdCesA8A protein during cellulose synthesis in aspen.

Molecular characterization of a cellulose synthase gene (AaxmCesA1) isolated from an Acacia auriculiformis x Acacia mangium hybrid.

Cellulose is the major component of plant cell walls, providing mechanical strength to the structural framework of plants. In association with lignin, hemicellulose, protein and pectin, cellulose forms the strong yet flexible bio-composite tissue of wood. Wood formation is an essential biological process and is of significant importance to the cellulosic private sector industry. Cellulose synthase genes encode the catalytic subunits of a large protein complex responsible for the biogenesis of cellulose in higher plants. The hybrid Acacia auriculiformis x Acacia mangium represents an important source of tree cellulose for forest-based product manufacturing, with enormous economic potential. In this work, we isolate the first cellulose synthase gene, designated AaxmCesA1, from this species. The isolated full-length AaxmCesA1 cDNA encodes a polypeptide of 1,064 amino acids. Sequence analyses revealed that AaxmCesA1 cDNA possesses the key motif characteristics of a CesA protein. AaxmCesA1 shares more than 75 % amino acid sequence identity with CesA proteins from other plant species. Subsequently, the full-length AaxmCesA1 gene of 7,389 bp with partial regulatory and 13 intron regions was also isolated. Relative gene expression analysis by quantitative PCR in different tissues of the Acacia hybrid, suggests the involvement of the AaxmCesA1 gene in primary cell wall synthesis of rapidly dividing young root cells. Similarity analyses using Blast algorithms also suggests a role in primary cell wall deposition in the Acacia hybrid. Southern analysis predicts that AaxmCesA1 is a member of a multigene family with at least two isoforms in the genome of the Acacia hybrid.

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

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