Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.     

Cellulose biosynthesis in plants: from genes to rosettes

Modern techniques of gene cloning have identified the CesA genes as encoding the probable catalytic subunits of the plant CelS, the cellulose synthase enzyme complex visualized in the plasma membrane as rosettes. At least 10 CesA isoforms exist in Arabidopsis and have been shown by mutant analyses to play distinct role/s in the cellulose synthesis process. Functional specialization within this family includes differences in gene expression, regulation and, possibly, catalytic function. Current data points towards some CesA isoforms potentially being responsible for initiation or elongation of the recently identified sterol beta-glucoside primer within different cell types, e.g. those undergoing either primary or secondary wall cellulose synthesis. Different CesA isoforms may also play distinct roles within the rosette, and there is some circumstantial evidence that CesA genes may encode the catalytic subunit of the mixed linkage glucan synthase or callose synthase. Various other proteins such as the Korrigan endocellulase, sucrose synthase, cytoskeletal components, Rac13, redox proteins and a lipid transfer protein have been implicated to be involved in synthesizing cellulose but, apart from CesAs, only Korrigan has been definitively linked with cellulose synthesis. These proteins should prove valuable in identifying additional CelS components.     

Identification of cellulose synthase AtCesA7 (IRX3) in vivo phosphorylation sites—a potential role in regulating protein degradation

Cellulose is central to plant development and is synthesised at the plasma membrane by an organised protein complex that contains three different cellulose synthase proteins. The ordered assembly of these three catalytic subunits is essential for normal cellulose synthesis. The way in which the relative levels of these three proteins are regulated within the cell is currently unknown. In this work it is shown that one of the cellulose synthases essential for secondary cell wall cellulose synthesis in Arabidopsis thaliana, AtCesA7, is phosphorylated in vivo. Analysis of in vivo phosphorylation sites by mass spectrometry reveals that two serine residues are phosphorylated. These residues occur in a region of hyper-variability between the cellulose synthase catalytic subunits. The region of the protein containing these phosphorylation sites can be phosphorylated by a plant extract in vitro. Incubation of this region with plant extracts results in its degradation via a proteasome dependant pathway. Full length endogenous CesA7 is also degraded via a proteasome dependant pathway in whole plant extracts. This data suggests that phosphorylation of the catalytic subunits may target them for degradation via a proteasome dependant pathway. This is a possible mechanism by which plants regulate the relative levels of the three proteins whose specific interaction are required to form an active cellulose synthase complex. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Higher plant cellulose synthases

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.     

Carbon partitioning to cellulose synthesis

This article discusses the importance and implications of regulating carbon partitioning to cellulose synthesis, the characteristics of cells that serve as major sinks for cellulose deposition, and enzymes that participate in the conversion of supplied carbon to cellulose. Cotton fibers, which deposit almost pure cellulose into their secondary cell walls, are referred to as a primary model system. For sucrose synthase, we discuss its proposed role in channeling UDP-Glc to cellulose synthase during secondary wall deposition, its gene family, its manipulation in transgenic plants, and mechanisms that may regulate its association with sites of polysaccharide synthesis. For cellulose synthase, we discuss the organization of the gene family and how protein diversity could relate to control of carbon partitioning to cellulose synthesis. Other enzymes emphasized include UDP-Glc pyrophosphorylase and sucrose phosphate synthase. New data are included on phosphorylation of cotton fiber sucrose synthase, possible regulation by Ca²⁺ of sucrose synthase localization, electron microscopic immunolocalization of sucrose synthase in cotton fibers, and phylogenetic relationships between cellulose synthase proteins, including three new ones identified in differentiating tracheary elements of Zinnia elegans. We develop a model for metabolism related to cellulose synthesis that implicates the changing intracellular localization of sucrose synthase as a molecular switch between survival metabolism and growth and/or differentiation processes involving cellulose synthesis. Abbreviations: CesA, cellulose synthase; Csl, cellulose-like synthase (genes); DCB, dichlobenil; DPA, days after anthesis; SPS, sucrose phosphate synthase; SuSy, sucrose synthase; P-SuSy, particulate SuSy; S-SuSy, soluble SuSy     

The Structure of the Catalytic Domain of a Plant Cellulose Synthase and Its Assembly into Dimers

Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.     

PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.     

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

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

Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall

Several brittle culm mutations of rice (Oryza sativa) causing fragility of plant tissues have been identified genetically but not characterized at a molecular level. We show here that the genes responsible for three distinct brittle mutations of rice, induced by the insertion of the endogenous retrotransposon Tos17, correspond to CesA (cellulose synthase catalytic subunit) genes, OsCesA4, OsCesA7 and OsCesA9. Three CesA genes were expressed in seedlings, culms, premature panicles, and roots but not in mature leaves, and the expression profiles were almost identical among the three genes. Cellulose contents were dramatically decreased (8.9%-25.5% of the wild-type level) in the culms of null mutants of the three genes, indicating that these genes are not functionally redundant. Consistent with these results, cell walls in the cortical fiber cells were shown to be thinner in all the mutants than in wild-type plants. Based on these observations, the structure of a cellulose-synthesizing complex involved in the synthesis of the secondary cell wall is discussed.     

The roles of the cytoskeleton during cellulose deposition at the secondary cell wall

During secondary cell wall formation, developing xylem vessels deposit cellulose at specific sites on the plasma membrane. Bands of cortical microtubules mark these sites and are believed to somehow orientate the cellulose synthase complexes. We have used live cell imaging on intact roots of Arabidopsis to explore the relationship between the microtubules, actin and the cellulose synthase complex during secondary cell wall formation. The cellulose synthase complexes are seen to form bands beneath sites of secondary wall synthesis. We find that their maintenance at these sites is dependent upon underlying bundles of microtubules which localize the cellulose synthase complex (CSC) to the edges of developing cell wall thickenings. Thick actin cables run along the long axis of the cells. These cables are essential for the rapid trafficking of complex-containing organelles around the cell. The CSCs appear to be delivered directly to sites of secondary cell wall synthesis and it is likely that transverse actin may mark these sites.     

A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region

Three membrane proteins required for cell division in Escherichia coli, FtsQ, FtsL and FtsB, localize to the cell septum. FtsL and FtsB, which each contain a leucine zipper-like sequence, are dependent on each other for this localization, and each of them is dependent on FtsQ. However, FtsQ is found at the cell division site in the absence of FtsL and FtsB. FtsQ, in turn, requires FtsK for its localization. Here, we show that FtsL, FtsB and FtsQ form a complex in vivo. Strikingly, this complex forms in the absence of FtsK, which is required for the localization of all three proteins to the mid-cell. These findings indicate that the FtsL, FtsB, FtsQ interactions can take place in cells before movement to the mid-cell and that migration to this position might occur only after the formation of the complex. Evidence indicating the regions of the three proteins involved in complex formation is presented. These findings provide the first example of preassembly of a subcomplex of cell division proteins before their localization to the septal region.     

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2 (AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1(-)) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3 were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31 degrees C.     

Transformation proteins and DNA uptake localize to the cell poles in Bacillus subtilis

The Gram-positive, rod-forming bacterium Bacillus subtilis efficiently binds and internalizes transforming DNA. The localization of several competence proteins, required for DNA uptake, has been studied using fluorescence microscopy. At least three proteins (ComGA, ComFA, and YwpH) are preferentially associated with the cell poles and appear to colocalize. This association is dynamic; the proteins accumulate at the poles as transformability develops and then delocalize as transformability wanes. DNA binding and uptake also occur preferentially at the cell poles, as shown using fluorescent DNA and in single-molecule experiments with laser tweezers. In addition to the prominent polar sites, the competence proteins also localize as foci in association with the lateral cell membrane, but this distribution does not exhibit the same temporal changes as the polar accumulation. The results suggest the regulated assembly and disassembly of a DNA-uptake machine at the cell poles.     

Cellulose synthase (CesA) genes in the green alga Mesotaenium caldariorum

Cellulose, a microfibrillar polysaccharide consisting of bundles of beta-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.     

Recent progress in cellulose biosynthesis

Cellulose comprises the major polymer of the plant cell wall. It consists of a set of parallel chains composed of glucans and these chains are highly oriented to form a structure known as a microfibril. The orientation of the microfibrils controls the extension of the direction of the plant cell. Extensive studies on the cellulose biosynthesis have been carried out for over three decades, and recently (1996) genes for cellulose biosynthesis in plants (CesA) were isolated. In the year 2002, a specific primer for cellulose biosynthesis reaction has been discovered and cellulose synthetic activity has been also confirmed by recombinant protein derived from the plant CesA gene. Furthermore, other proteins involved in cellulose biosynthesis besides CesA proteins were also proposed at the same time. One of these proteins, Korrigan cellulase, was suggested to act by removing sitosterol from the primer for biosynthesis reaction of cellulose. A membrane-bound sucrose synthase was also suggested to provide UDP-glucose as a substrate for cellulose biosynthesis. On the basis of these results, a new pathway for cellulose biosynthesis was proposed. Now, the research field of cellulose biosynthesis is facing a major turning point. Electronic Publication