CesA Proteins Research Articles

Cellulose Synthesis in Higher Plants

Cellulose microfibrils play essential roles in the organization of plant cell walls, thereby allowing a growth habit based on turgor. The fibrils are made by 30 nm diameter plasma membrane complexes composed of approximately 36 subunits representing at least three types of related CESA proteins. The complexes assemble in the Golgi, where they are inactive, and move to the plasma membrane, where they become activated. The complexes move through the plasma membrane during cellulose synthesis in directions that coincide with the orientation of microtubules. Recent, simultaneous, live-cell imaging of cellulose synthase and microtubules indicates that the microtubules exert a direct influence on the orientation of cellulose deposition. Genetic studies in Arabidopsis have identified a number of genes that contribute to the overall process of cellulose synthesis, but the role of these proteins is not yet known.

The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo.

Little is known about the assembly and turnover of cellulose synthase complexes commonly called rosettes. Recent work indicates that rosette assembly could involve the dimerization of CesA (cellulose synthase catalytic subunit) proteins regulated by the redox state of the CesA zinc-binding domain (ZnBD). Several studies in the 1980s led to the suggestion that synthase complexes may have very short half-lives in vivo, but no recent work has directly addressed this issue. In the present work, we show that the half-life of cotton fiber GhCesA1 protein is <30 min in vivo, far less than the average membrane protein. We also show that the reduced monomer of GhCesA1 ZnBD is rapidly degraded when exposed to cotton fiber extracts, whereas the oxidized dimer is resistant to degradation. Low rates of degradation activity were detected in vitro by using extracts from fibers harvested during primary cell-wall formation, but activity increased markedly during transition to secondary cell-wall synthesis. In vitro degradation of reduced GhCesA1 ZnBD is inhibited by proteosome inhibitor MG132 and also by E64 and EGTA, suggesting that proteolysis is initiated by cysteine protease activity rather than the proteosome. We used a yeast two-hybrid system to identify a putative cotton fiber metallothionein and to confirm it as a protein that could interact with the GhCesA1 ZnBD. A model is proposed wherein active cellulose synthase complexes contain CesA proteins in dimerized form, and turnover and degradation of the complexes are mediated through reductive zinc insertion by metallothionein and subsequent proteolysis involving a cysteine protease.

Six new cellulose synthase genes from Eucalyptus are associated with primary and secondary cell wall biosynthesis

Higher plants contain a family of cellulose synthase catalytic subunit (CesA) genes that encode components of an enzyme complex embedded in the cell membrane. Recent studies in several higher plant species have demonstrated that two groups of CesA genes exist, associated with either primary or secondary cell wall deposition. We cloned six full-length CesA cDNAs from Eucalyptus grandis W. Hill ex Maiden (EgCesA1 through 6) and determined their expression patterns in a variety of organs from an adult tree. The six EgCesA genes encode predicted proteins of 978 to 1097 amino acid residues, each of which contains all of the key regions and motifs characteristic of functional CESA proteins. The predicted proteins share limited amino acid identity with each other, ranging from 61 to 70%. In contrast, similar CESA proteins from higher plant species exhibit 81 to 90% identity with the six EgCESAs. Gene expression analysis using quantitative reverse-transcription polymerase chain reaction indicated that transcripts of EgCesA1 to 3 were abundant in tissues enriched with cells laying down secondary cell walls (e.g., xylem), but were weakly expressed in tissues undergoing primary growth (e.g., unfolding leaves). Expression of EgCesA4 and EgCesA5 was upregulated in tissues rich in rapidly dividing cells undergoing primary wall synthesis, whereas EgCesA6 was weakly expressed in all tissues analyzed. These results suggest that Eucalyptus, like other higher plants, expresses two contrasting groups of apparently co-regulated CesAs involved in either primary or secondary cell wall biosynthesis.

The pivotal role of cyanobacteria in the evolution of cellulose synthases and cellulose synthase-like proteins

Cellulose synthase and other members of the family 2 glycosyltransferases are ubiquitous in all kingdoms of life. To date, no attempt has been made to construct a phylogeny that positions cellulose synthases in relation to other members of this family or to elucidate relationships within the cellulose synthase group. In this study, a sequence from the unicellular, marine cyanobacterium Synechococcus sp. PCC 7002 is shown to share a unique common ancestor of a clade consisting of cellulose synthases from Dictyostelium discoideum and Nostoc, as well as a plant grouping that includes CesA proteins and cellulose synthase-like (Csl) proteins G, E, B, D, and F. A branching order is established for Csl proteins that places CslG as ancestral to other members of the Csl/CesA clade. Sequences from Ciona intestinalis and Aspergillis fumigatus are shown to branch at the base of the Eukaryota/Cyanobacteria clade. These data suggest multiple independent transfers of cellulose synthases. The implications of these findings in relation to the evolutionary history of cellulose synthase are discussed.

Cloning and characterization of cellulose synthase‐like gene, PtrCSLD2 from developing xylem of aspen trees

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.

Control of cellulose synthase complex localization in developing xylem.

Cellulose synthesis in the developing xylem vessels of Arabidopsis requires three members of the cellulose synthase (CesA) gene family. In young vessels, these three proteins localize within the cell, whereas in older vessels, all three CesA proteins colocalize with bands of cortical microtubules that mark the sites of secondary cell wall deposition. In the absence of one subunit, however, the remaining two subunits are retained in the cell, demonstrating that all three CesA proteins are required to assemble a functional complex. CesA proteins with altered catalytic activity localize normally, suggesting that cellulose synthase activity is not required for this localization. Cortical microtubule arrays are required continually to maintain normal CesA protein localization. By contrast, actin microfilaments do not colocalize with the CesA proteins and are unlikely to play a direct role in their localization. Green fluorescent protein-tagged CesA reveals a novel process in which the structure and/or local environment of the cellulose synthase complex is altered rapidly.

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.

Interactions among three distinct CesA proteins essential for cellulose synthesis.

In a screen to identify novel cellulose deficient mutants, three lines were shown to be allelic and define a novel complementation group, irregular xylem5 (irx5). IRX5 was cloned and encodes a member of the CesA family of cellulose synthase catalytic subunits (AtCesA4). irx5 plants have an identical phenotype to previously described mutations in two other members of this gene family (IRX1 and IRX3). IRX5, IRX3, and IRX1 are coexpressed in exactly the same cells, and all three proteins interact in detergent solubilized extracts, suggesting that three members of this gene family are required for cellulose synthesis in secondary cell walls. The association of IRX1 and IRX3 was reduced to undetectable levels in the absence of IRX5. Consequently, these data suggest that IRX5, IRX3, and IRX1 are all essential components of the cellulose synthesizing complex and the presence of all three subunits is required for the correct assembly of this complex.

Xylem-Specific and Tension Stress-Responsive Expression of Cellulose Synthase Genes from Aspen Trees

Genetic improvement of cellulose biosynthesis in woody trees is one of the major goals of tree biotechnology research. Yet, progress in this field has been slow owing to (1) unavailability of key genes from tree genomes, (2) the inability to isolate active and intact cellulose synthase complexes and, (3) the limited understanding of the mechanistic processes involved in the wood cellulose development. Here I report on the recent advances in molecular genetics of cellulose synthases (CesA) from aspen trees. Two different types of cellulose synthases appear to be involved in cellulose deposition in primary and secondary walls in aspen xylem. The three distinct secondary CesAs from aspen- PtrCesA1, PtrCesA2, and PtrCesA3-appear to be aspen homologs of Arabidopsis secondary CesAs AtCesA8, AtCesA7, and AtCesA4, respectively, based on their high identity/similarity (>80%). These aspen CesA proteins share the transmembrane domain (TMD) structure that is typical of all known "true" CesA proteins: two TMDs toward the N-terminal and six TMDs toward the C-terminal. The putative catalytic domain is present between TMDs 2 and 3. All signature motifs of processive glycosyltransferases are also present in this catalytic domain. In a phylogenetic tree based on various predicted CesA proteins from Arabidopsis and aspen, aspen CesAs fall into families similar to those seen with Arabidopsis CesAs, suggesting their functional similarity. The coordinate expression of three aspen secondary CesAs in xylem and phloem fibers, along with their simultaneous tension stress-responsive upregulation, suggests that these three CesAs may play a pivotal role in biosynthesis of better-quality cellulose in secondary cell walls of plants. These results are likely to have a direct impact on genetic manipulation of trees in the future.

Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains.

Cellulose synthase (CesA) proteins are components of CesA complexes (rosettes) and are thought to catalyze the chain elongation step in glucan polymerization. Little is understood about rosette assembly, including how CesAs interact with each other or with other components within the complexes. The first conserved region at the N terminus of plant CesA proteins contains two putative zinc fingers that show high homology to the RING-finger motif. We show that this domain in GhCesA1 can bind two atoms of Zn2+, as predicted by its structure. Analysis in the yeast two-hybrid system indicates that the N-terminal portions of cotton fiber GhCesA1 and GhCesA2 containing these domains can interact to form homo- or heterodimers. Although Zn(2+) binding occurs only when the protein is in the reduced form, biochemical analyses show that under oxidative conditions, the GhCesA1 zinc-finger domain and also the full-length protein dimerize via intermolecular disulfide bonds, indicating CesA dimerization can be regulated by redox state. We also provide evidence that the herbicide CGA 325'615 (Syngenta, Basel), which inhibits synthesis of crystalline cellulose and leads to a disruption of rosette architecture, may affect the oxidative state of the zinc-finger domain that is necessary for rosette stability. Taken together, these results support a model in which at least part of the process of rosette assembly and function may involve oxidative dimerization between CesA subunits.

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.

The experimental herbicide CGA 325'615 inhibits synthesis of crystalline cellulose and causes accumulation of non-crystalline beta-1,4-glucan associated with CesA protein.

Developing cotton (Gossypium hirsutum) fibers, cultured in vitro with their associated ovules, were used to compare the effects of two herbicides that inhibit cellulose synthesis: 2,6-dichlorobenzonitrile (DCB) and an experimental thiatriazine-based herbicide, CGA 325'615. CGA 325'615 in nanomolar concentrations or DCB in micromolar concentrations causes inhibition of synthesis of crystalline cellulose. Unlike DCB, CGA 325'615 also causes concomitant accumulation of non-crystalline beta-1,4-glucan that can be at least partially solubilized from fiber walls with ammonium oxalate. The unusual solubility of this accumulated glucan may be explained by its strong association with protein. Treatment of the glucan fraction with protease changes its size distribution and leads to precipitation of the glucan. Treatment of the glucan fraction with cellulase digests the glucan and also releases protein that has been characterized as GhCesA-1 and GhCesA-2--proteins that are believed to represent the catalytic subunit of cellulose synthase. The fact that cellulase treatment is required to release this protein indicates an extremely tight association of the glucan with the CesA proteins. In addition, CGA 325'615, but not DCB, also causes accumulation of CesA protein and a membrane-associated cellulase in the membrane fraction of fibers. In addition to the effects of CGA 325'615 on levels of both of these proteins, the level of both also shows coordinate regulation during fiber development, further suggesting they are both important for cellulose synthesis. The accumulation of non-crystalline glucan caused by CGA 325'615 mimics the phenotype of the cellulose-deficient rsw1 mutant of Arabidopsis that also accumulates an apparently similar glucan (T. Arioli, L. Peng, A.S. Betzner, J. Burn, W. Wittke, W. Herth, C. Camilleri, H. Hofte, J. Plazinski, R. Birch et al. [1998] Science 279: 717).