Endosidin20-1 is more potent than endosidin20 in inhibiting plant cellulose biosynthesis and molecular docking analysis of cellulose biosynthesis inhibitors on modeled cellulose synthase structure

Endosidin20 (ES20) is a recently identified cellulose biosynthesis inhibitor (CBI) that targets the catalytic site of plant cellulose synthase (CESA). Here, we screened over 600 ES20 analogs and identified nine active analogs named ES20-1 to ES20-9. Among these, endosidin20-1 (ES20-1) had stronger inhibitory effects on plant growth and cellulose biosynthesis than ES20. At the biochemical level, we demonstrated that ES20-1, like ES20, directly interacts with CESA6. At the cellular level, this molecule, like ES20, induced the accumulation of cellulose synthase complexes at the Golgi apparatus and inhibited their secretion to the plasma membrane. Like ES20, ES20-1 likely targets the catalytic site of CESA. However, through molecular docking analysis using a modeled structure of full-length CESA6, we found that both ES20 and ES20-1 might have another target site at the transmembrane regions of CESA6. Besides ES20, other CBIs such as isoxaben, C17, and flupoxam are widely used tools to dissect the mechanism of cellulose biosynthesis and are also valuable resources for the development of herbicides. Here, based on mutant genetic analysis and molecular docking analysis, we have identified the potential target sites of these CBIs on a modeled CESA structure. Some bacteria also produce cellulose, and both ES20 and ES20-1 inhibited bacterial cellulose biosynthesis. Therefore, we conclude that ES20-1 is a more potent analog of ES20 that inhibits intrinsic cellulose biosynthesis in plants, and both ES20 and ES20-1 show an inhibitory effect on bacterial growth and cellulose synthesis, making them excellent tools for exploring the mechanisms of cellulose biosynthesis across kingdoms.     

Trans-Golgi Network—An Intersection of Trafficking Cell Wall Components

The cell wall, a crucial cell compartment, is composed of a network of polysaccharides and proteins, providing structural support and protection from external stimuli. While the cell wall structure and biosynthesis have been extensively studied, very little is known about the transport of polysaccharides and other components into the developing cell wall. This review focuses on endomembrane trafficking pathways involved in cell wall deposition. Cellulose synthase complexes are assembled in the Golgi, and are transported in vesicles to the plasma membrane. Non-cellulosic polysaccharides are synthesized in the Golgi apparatus, whereas cellulose is produced by enzyme complexes at the plasma membrane. Polysaccharides and enzymes that are involved in cell wall modification and assembly are transported by distinct vesicle types to their destinations; however, the precise mechanisms involved in selection, sorting and delivery remain to be identified. The endomembrane system orchestrates the delivery of Golgi-derived and possibly endocytic vesicles carrying cell wall and cell membrane components to the newly-formed cell plate. However, the nature of these vesicles, their membrane compositions, and the timing of their delivery are largely unknown. Emerging technologies such as chemical genomics and proteomics are promising avenues to gain insight into the trafficking of cell wall components.     

The trafficking of the cellulose synthase complex in higher plants

Background

Cellulose is an important constituent of plant cell walls in a biological context, and is also a material commonly utilized by mankind in the pulp and paper, timber, textile and biofuel industries. The biosynthesis of cellulose in higher plants is a function of the cellulose synthase complex (CSC). The CSC, a large transmembrane complex containing multiple cellulose synthase proteins, is believed to be assembled in the Golgi apparatus, but is thought only to synthesize cellulose when it is localized at the plasma membrane, where CSCs synthesize and extrude cellulose directly into the plant cell wall. Therefore, the delivery and endocytosis of CSCs to and from the plasma membrane are important aspects for the regulation of cellulose biosynthesis.

Scope

Recent progress in the visualization of CSC dynamics in living plant cells has begun to reveal some of the routes and factors involved in CSC trafficking. This review highlights the most recent major findings related to CSC trafficking, provides novel perspectives on how CSC trafficking can influence the cell wall, and proposes potential avenues for future exploration.     

Membrane trafficking mediated by OsDRP2B is specific for cellulose biosynthesis

Increasing evidence has revealed that membrane trafficking is highly associated with cell wall metabolism. Factors involved in vesicle delivery, e.g., cytoskeleton and motor proteins, have showed regulatory effects on cell wall structure and components. However, little is known about the involvement of other trafficking components in distribution of cell wall-related compartments. Dynamins are important proteins functioning in membrane tubulation and vesiculation. Recently, we have reported characterization of the rice dynamin-related protein 2B (OsDRP2B). Mutation in OsDRP2B causes a significant reduction in cellulose content. Its association with the trans-Golgi network (TGN) and clathrin-coated vesicles and the reduced CESA4 abundance at the bc3 plasma membrane suggest that BC3/OsDRP2B is involved in the transport of essential elements for cellulose synthesis. Here, we provide additional evidence for BC3 subcellular localization via observing OsDRP2B-GFP in living root hairs of transgenic plants. Uronic acid and fractional composition analyses further confirm that the amount of arabinoxylan and other noncellulosic polysaccharides is increased in bc3. However, three putative xylan synthesis genes are downregulated in mutant plant revealed by real-time PCR analysis. These results imply that compartments delivered by OsDRP2B are specifically responsible for cellulose biosynthesis.Key words: OsDRP2B, cellulose biosynthesis, membrane trafficking, brittleness, ricePlant cell wall is an extracellular matrix enriched in polysaccharides. Except for cellulose that is produced at the plasma membrane by cellulose synthase (CESA) complexes, most of the cell wall products are assumed being synthesized inside cells, e.g., in the Golgi apparatus and secreted outside through complex membrane trafficking. Besides the cell wall-localized products, some proteins essential for cellulose biosynthesis need to be translocated onto the plasma membrane to facilitate cellulose formation.^1,2 Intracellular trafficking is therefore a key level for regulating cell wall composition and architecture, which are highly dynamic during cellular development.³ This notion is substantiated by the fact that wall architecture within the same cell is heterogeneity, indicating the presence of cell wall specific deposition domains.^4,5 For example, pectins are often located at the cell corners.³ Different de-esterified homogalacturonan (HG) are present along the growing pollen tubes or root hairs: tips have highly esterified HG; the de-esterified degree is increased after tips.⁶ Although it is believed that these specific patterns could be the result of the targeted secretion of polysaccharides,³ our knowledge about the polysaccharide secretion is still very few. Currently, in vivo viewing CESA-containing compartments and the movement inside living cells have provided direct evidence for the trafficking action of CESA compartments.^2,7,8 The delivery and removal of CESA complexes to/from the plasma membrane are very complicated, which require the involvement of many components, such as cytoskeleton and syntaxins.^7,9,10 Syntaxins, part of SNARE complexes, function as docking factor of cell wall-related compartments during cell plate formation.¹⁰ Dynamin and dynamin-related proteins (DRPs) are involved in diverse events of cellular membrane remodeling.¹¹ It remains unknown about whether DRPs are responsible for CESA trafficking. Recently, we have reported that BC3, the rice DRP2B protein, plays a role in complex membrane trafficking and affects the biosynthesis of secondary walls. Here, we provide additional cellular and wall chemical data to confirm that BC3/OsDRP2B is specifically involved in the secondary cell wall cellulose synthesis.     

Chitinase-like1/pom-pom1 and its homolog CTL2 are glucan-interacting proteins important for cellulose biosynthesis in Arabidopsis

Plant cells are encased by a cellulose-containing wall that is essential for plant morphogenesis. Cellulose consists of β-1,4-linked glucan chains assembled into paracrystalline microfibrils that are synthesized by plasma membrane-located cellulose synthase (CESA) complexes. Associations with hemicelluloses are important for microfibril spacing and for maintaining cell wall tensile strength. Several components associated with cellulose synthesis have been identified; however, the biological functions for many of them remain elusive. We show that the chitinase-like (CTL) proteins, CTL1/POM1 and CTL2, are functionally equivalent, affect cellulose biosynthesis, and are likely to play a key role in establishing interactions between cellulose microfibrils and hemicelluloses. CTL1/POM1 coincided with CESAs in the endomembrane system and was secreted to the apoplast. The movement of CESAs was compromised in ctl1/pom1 mutant seedlings, and the cellulose content and xyloglucan structures were altered. X-ray analysis revealed reduced crystalline cellulose content in ctl1 ctl2 double mutants, suggesting that the CTLs cooperatively affect assembly of the glucan chains, which may affect interactions between hemicelluloses and cellulose. Consistent with this hypothesis, both CTLs bound glucan-based polymers in vitro. We propose that the apoplastic CTLs regulate cellulose assembly and interaction with hemicelluloses via binding to emerging cellulose microfibrils.     

CELLULOSE SYNTHASE INTERACTIVE1 Is Required for Fast Recycling of Cellulose Synthase Complexes to the Plasma Membrane in Arabidopsis

Plants are constantly subjected to various biotic and abiotic stresses and have evolved complex strategies to cope with these stresses. For example, plant cells endocytose plasma membrane material under stress and subsequently recycle it back when the stress conditions are relieved. Cellulose biosynthesis is a tightly regulated process that is performed by plasma membrane-localized cellulose synthase (CESA) complexes (CSCs). However, the regulatory mechanism of cellulose biosynthesis under abiotic stress has not been well explored. In this study, we show that small CESA compartments (SmaCCs) or microtubule-associated cellulose synthase compartments (MASCs) are critical for fast recovery of CSCs to the plasma membrane after stress is relieved in Arabidopsis thaliana. This SmaCC/MASC-mediated fast recovery of CSCs is dependent on CELLULOSE SYNTHASE INTERACTIVE1 (CSI1), a protein previously known to represent the link between CSCs and cortical microtubules. Independently, AP2M, a core component in clathrin-mediated endocytosis, plays a role in the formation of SmaCCs/MASCs. Together, our study establishes a model in which CSI1-dependent SmaCCs/MASCs are formed through a process that involves endocytosis, which represents an important mechanism for plants to quickly regulate cellulose synthesis under abiotic stress.     

A missense mutation in the transmembrane domain of CESA4 affects protein abundance in the plasma membrane and results in abnormal cell wall biosynthesis in rice

Cellulose synthase (CESA) is a critical catalytic subunit of the cellulose synthase complex responsible for glucan chain elongation. Our knowledge about how CESA functions is still very limited. Here, we report the functional characterization of a rice mutant, brittle culm11, that shows growth retardation and dramatically reduced plant strength. Map-based cloning revealed that all the mutant phenotypes result from a missense mutation in OsCESA4 (G858R), a highly conserved residue at the end of the fifth transmembrane domain. The aberrant secondary cell wall of the mutant plants is attributed to significantly reduced cellulose content, abnormal secondary wall structure of sclerenchyma cells, and overall altered wall composition, as detected by chemical analyses and immunochemical staining. Importantly, we have found that this point mutation decreases the abundance of OsCESA4 in the plasma membrane, probably due to a defect in the process of CESA complex secretion. The data from our biochemical, genetic, and pharmacological analyses indicate that this residue is critical for maintaining the normal level of CESA proteins in the plasma membrane.     

The POK/AtVPS52 protein localizes to several distinct post-Golgi compartments in sporophytic and gametophytic cells

The organization and dynamics of the plant endomembrane system require both universal and plant-specific molecules and compartments. The latter, despite the growing wealth of information, remains poorly understood. From the study of an Arabidopsis thaliana male gametophytic mutant, it was possible to isolate a gene named POKY POLLEN TUBE (POK) essential for pollen tube tip growth. The similarity between the predicted POK protein sequence and yeast Vps52p, a subunit from the GARP/VFT complex which is involved in the docking of vesicles from the prevacuolar compartment to the Golgi apparatus, suggested that the POK protein plays a role in plant membrane trafficking. Genetic analysis of Arabidopsis mutants affecting AtVPS53 or AtVPS54 genes which encode putative POK partners shows a transmission defect through the male gametophyte for all lines, which is similar to the pok mutant. Using a combination of biochemical approaches and specific antiserum it has been demonstrated that the POK protein is present in phylogenetically divergent plant species, associated with membranes and belongs to a high molecular weight complex. Combination of immunolocalization studies and pharmacological approaches in different plant cells revealed that the POK protein associates with Golgi and post-Golgi compartments. The role of POK in post-Golgi endomembrane trafficking and as a member of a putative plant GARP/VFT complex is discussed.     

The plant secretory pathway seen through the lens of the cell wall

Secretion in plant cells is often studied by looking at well-characterised, evolutionarily conserved membrane proteins associated with particular endomembrane compartments. Studies using live cell microscopy and fluorescent proteins have illuminated the highly dynamic nature of trafficking, and electron microscopy studies have resolved the ultrastructure of many compartments. Biochemical and molecular analyses have further informed about the function of particular proteins and endomembrane compartments. In plants, there are over 40 cell types, each with highly specialised functions, and hence potential variations in cell biological processes and cell wall structure. As the primary function of secretion in plant cells is for the biosynthesis of cell wall polysaccharides and apoplastic transport complexes, it follows that utilising our knowledge of cell wall glycosyltransferases (GTs) and their polysaccharide products will inform us about secretion. Indeed, this knowledge has led to novel insights into the secretory pathway, including previously unseen post-TGN secretory compartments. Conversely, our knowledge of trafficking routes of secretion will inform us about polarised and localised deposition of cell walls and their constituent polysaccharides/glycoproteins. In this review, we look at what is known about cell wall biosynthesis and the secretory pathway and how the different approaches can be used in a complementary manner to study secretion and provide novel insights into these processes.     

Cellulose synthase interactive protein 1 (CSI1) mediates the intimate relationship between cellulose microfibrils and cortical microtubules

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.     

Resistance against herbicide isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6

Isoxaben is a pre-emergence herbicide that inhibits cellulose biosynthesis in higher plants. Two loci identified by isoxaben-resistant mutants (ixr1-1, ixr1-2, and ixr2-1) in Arabidopsis have been reported previously. IXR1 was recently shown to encode the cellulose synthase catalytic subunit CESA3 (W.-R. Scheible, R. Eshed, T. Richmond, D. Delmer, and C. Somerville [2001] Proc Natl Acad Sci USA 98: 10079-10084). Here, we report on the cloning of IXR2, and show that it encodes another cellulose synthase isoform, CESA6. ixr2-1 carries a mutation substituting an amino acid close to the C terminus of CESA6 that is highly conserved among CESA family members. Transformation of wild-type plants with the mutated gene and not with the wild-type gene conferred increased resistance against the herbicide. The simplest interpretation for the existence of these two isoxaben-resistant loci is that CESA3 and CESA6 have redundant functions. However, loss of function procuste1 alleles of CESA6 were previously shown to have a strong growth defect and reduced cellulose content in roots and dark-grown hypocotyls. This indicates that in these mutants, the presence of CESA3 does not compensate for the absence of CESA6 in roots and dark-grown hypocotyls, which argues against redundant functions for CESA3 and CESA6. Together, these observations are compatible with a model in which CESA6 and CESA3 are active as a protein complex.     

Functional analysis of complexes with mixed primary and secondary cellulose synthases

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 (irx1^cesa8, irx3^cesa7 and irx5^cesa4). 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 irx1^cesa8 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.     

Cellulose synthesis: a complex complex

Cellulose is the world's most abundant biopolymer and a key structural component of the plant cell wall. Cellulose is comprised of hydrogen-bonded beta-1,4-linked glucan chains that are synthesized at the plasma membrane by large cellulose synthase (CESA) complexes. Recent advances in visualization of fluorescently labelled complexes have facilitated exploration of regulatory modes of cellulose production. For example, several herbicides, such as isoxaben and 2,6-dichlorobenzonitrile that inhibit cellulose production appear to affect different aspects of synthesis. Dual-labelling of cytoskeletal components and CESAs has revealed dynamic feedback regulation between cellulose synthesis and microtubule orientation and organization. In addition, fluorescently tagged CESA2 subunits may substitute for another subunit, CESA6, which suggests both plasticity and specificity for one of the components of the CESA complex.     

Pausing of Golgi Bodies on Microtubules Regulates Secretion of Cellulose Synthase Complexes in Arabidopsis

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by plasma membrane–bound complexes containing cellulose synthase proteins (CESAs). Here, we establish a role for the cytoskeleton in intracellular trafficking of cellulose synthase complexes (CSCs) through the in vivo study of the green fluorescent protein (GFP)-CESA3 fusion protein in Arabidopsis thaliana hypocotyls. GFP-CESA3 localizes to the plasma membrane, Golgi apparatus, a compartment identified by the VHA-a1 marker, and, surprisingly, a novel microtubule-associated cellulose synthase compartment (MASC) whose formation and movement depend on the dynamic cortical microtubule array. Osmotic stress or treatment with the cellulose synthesis inhibitor CGA 325''615 induces internalization of CSCs in MASCs, mimicking the intracellular distribution of CSCs in nongrowing cells. Our results indicate that cellulose synthesis is coordinated with growth status and regulated in part through CSC internalization. We find that CSC insertion in the plasma membrane is regulated by pauses of the Golgi apparatus along cortical microtubules. Our data support a model in which cortical microtubules not only guide the trajectories of CSCs in the plasma membrane, but also regulate the insertion and internalization of CSCs, thus allowing dynamic remodeling of CSC secretion during cell expansion and differentiation.     

The rice dynamin‐related protein DRP2B mediates membrane trafficking,and thereby plays a critical role in secondary cell wall cellulose biosynthesis

Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin‐related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane‐associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence‐tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin‐mediated vesicles, but also is targeted to the trans‐Golgi network (TGN). An FM4‐64 uptake assay in transgenic plants that express green fluorescent protein‐tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post‐Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants.     

Phytochrome regulation of cellulose synthesis in Arabidopsis

Plant development is highly plastic and dependent on light quantity and quality monitored by specific photoreceptors. Although we have a detailed knowledge of light signaling pathways, little is known about downstream targets involved in growth control. Cell size and shape are in part controlled by cellulose microfibrils extruded from large cellulose synthase complexes (CSCs) that migrate in the plasma membrane along cortical microtubules. Here we show a role for the red/far-red light photoreceptor PHYTOCHROME B (PHYB) in the regulation of cellulose synthesis in the growing Arabidopsis hypocotyl. In this organ, CSCs contains three distinct cellulose synthase (CESA) isoform classes: nonredundant CESA1 and CESA3 and a third class represented by partially redundant CESA2, CESA5, and CESA6. Interestingly, in the dark, depending on which CESA subunits occupy the third position, CSC velocity is more or less inhibited through an interaction with microtubules. Activation of PHYB overrules this inhibition. The analysis of cesa5 mutants shows a role for phosphorylation in the control of CSC velocity. These results, combined with the cesa5 mutant phenotype, suggest that cellulose synthesis is fine tuned through the regulated interaction of CSCs with microtubules and that PHYB signaling impinges on this process to maintain cell wall strength and growth in changing environments.     

Biosynthesis of plant cell wall polysaccharides - a complex process

Cellulose, a major component of plant cell walls, is made by dynamic complexes that move within the plasma membrane while depositing cellulose directly into the wall. On the other hand, matrix polysaccharides are made in the Golgi and delivered to the wall via secretory vesicles. Several Golgi proteins that are involved in glucomannan and xyloglucan biosynthesis have been identified, including some glycan synthases that show sequence similarity to the cellulose synthase proteins and several glycosytransferases that add sidechains to the polysaccharide backbones. Recent progress in identifying the proteins needed for polysaccharide biosynthesis should lead to an improved understanding of the molecular details of these complex processes, and eventually to an ability to manipulate them in an effort to generate plants that have improved properties for human uses.     

An integrative analysis of four CESA isoforms specific for fiber cellulose production between Gossypium hirsutum and Gossypium barbadense

Cotton fiber is an excellent model system of cellulose biosynthesis; however, it has not been widely studied due to the lack of information about the cellulose synthase (CESA) family of genes in cotton. In this study, we initially identified six full-length CESA genes designated as GhCESA5–GhCESA10. Phylogenetic analysis and gene co-expression profiling revealed that CESA1, CESA2, CESA7, and CESA8 were the major isoforms for secondary cell wall biosynthesis, whereas CESA3, CESA5, CESA6, CESA9, and CESA10 should involve in primary cell wall formation for cotton fiber initiation and elongation. Using integrative analysis of gene expression patterns, CESA protein levels, and cellulose biosynthesis in vivo, we detected that CESA8 could play an enhancing role for rapid and massive cellulose accumulation in Gossypium hirsutum and Gossypium barbadense. We found that CESA2 displayed a major expression in non-fiber tissues and that CESA1, a housekeeping gene like, was predominantly expressed in all tissues. Further, a dynamic alteration was observed in cell wall composition and a significant discrepancy was observed between the cotton species during fiber elongation, suggesting that pectin accumulation and xyloglucan reduction might contribute to cell wall transition. In addition, we discussed that callose synthesis might be regulated in vivo for massive cellulose production during active secondary cell wall biosynthesis in cotton fibers.