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.     

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.     

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.     

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.     

The N‐terminal zinc finger of CELLULOSE SYNTHASE6 is critical in defining its functional properties by determining the level of homodimerization in Arabidopsis

Primary cell wall cellulose is synthesized by the cellulose synthase complex (CSC) containing CELLULOSE SYNTHASE1 (CESA1), CESA3 and one of four CESA6‐like proteins in Arabidopsis. It has been proposed that the CESA6‐like proteins occupy the same position in the CSC, but their underlying selection mechanism remains unclear. We produced a chimeric CESA5 by replacing its N‐terminal zinc finger with its CESA6 counterpart to investigate the consequences for its homodimerization, a crucial step in forming higher‐order structures during assembly of the CSC. We found that the mutant phenotypes of prc1‐1, a cesa6 null mutant, were rescued by the chimeric CESA5, and became comparable to the wild type (WT) and prc1‐1 complemented by WT CESA6 in regard to plant growth, cellulose content, cellulose microfibril organization, CSC dynamics and subcellular localization. Bimolecular fluorescence complementation assays were employed to evaluate pairwise interactions between the N‐terminal regions of CESA1, CESA3, CESA5, CESA6 and the chimeric CESA5. We verified that the chimeric CESA5 explicitly interacted with all the other CESA partners, comparable to CESA6, whereas interaction between CESA5 with itself was significantly weaker than that of all other CESA pairs. Our findings suggest that the homodimerization of CESA6 through its N‐terminal zinc finger is critical in defining its functional properties, and possibly determines its intrinsic roles in facilitating higher‐order structures in CSCs.     

Subfunctionalization of cellulose synthases in seed coat epidermal cells mediates secondary radial wall synthesis and mucilage attachment

Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology.     

Rab-H_1b is essential for trafficking of cellulose synthase and for hypocotyl growth in Arabidopsis thaliana

Cell-wall deposition of cellulose microfibrils is essential for plant growth and development. In plant cells,cellulose synthesis is accomplished by cellulose synthase complexes located in the plasma membrane. Trafficking of the complex between endomembrane compartments and the plasma membrane is vital for cellulose biosynthesis;however, the mechanism for this process is not well understood. We here report that, in Arabidopsis thaliana,Rab-H_1b, a Golgi-localized small GTPase, participates in the trafficking of CELLULOSE SYNTHASE 6(CESA6) to the plasma membrane. Loss of Rab-H_1b function resulted in altered distribution and motility of CESA6 in the plasma membrane and reduced cellulose content. Seedlings with this defect exhibited short, fragile etiolated hypocotyls.Exocytosis of CESA6 was impaired in rab-h1 b cells, and endocytosis in mutant cells was significantly reduced as well. We further observed accumulation of vesicles around an abnormal Golgi apparatus having an increased number of cisternae in rab-h1 b cells, suggesting a defect in cisternal homeostasis caused by Rab-H_1b loss function. Our findings link Rab GTPases to cellulose biosynthesis, during hypocotyl growth, and suggest Rab-H_1b is crucial for modulating the trafficking of cellulose synthase complexes between endomembrane compartments and the plasma membrane and for maintaining Golgi organization and morphology.e     

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.     

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.     

OsCESA9 conserved‐site mutation leads to largely enhanced plant lodging resistance and biomass enzymatic saccharification by reducing cellulose DP and crystallinity in rice

Genetic modification of plant cell walls has been posed to reduce lignocellulose recalcitrance for enhancing biomass saccharification. Since cellulose synthase (CESA) gene was first identified, several dozen CESA mutants have been reported, but almost all mutants exhibit the defective phenotypes in plant growth and development. In this study, the rice (Oryza sativa) Osfc16 mutant with substitutions (W481C, P482S) at P‐CR conserved site in CESA9 shows a slightly affected plant growth and higher biomass yield by 25%–41% compared with wild type (Nipponbare, a japonica variety). Chemical and ultrastructural analyses indicate that Osfc16 has a significantly reduced cellulose crystallinity (CrI) and thinner secondary cell walls compared with wild type. CESA co‐IP detection, together with implementations of a proteasome inhibitor (MG132) and two distinct cellulose inhibitors (Calcofluor, CGA), shows that CESA9 mutation could affect integrity of CESA4/7/9 complexes, which may lead to rapid CESA proteasome degradation for low‐DP cellulose biosynthesis. These may reduce cellulose CrI, which improves plant lodging resistance, a major and integrated agronomic trait on plant growth and grain production, and enhances biomass enzymatic saccharification by up to 2.3‐fold and ethanol productivity by 34%–42%. This study has for the first time reported a direct modification for the low‐DP cellulose production that has broad applications in biomass industries.     

Genetic modification in cellulose‐synthase reduces crystallinity and improves biochemical conversion to fermentable sugar

The cellulose synthase (CESA) membrane complex synthesizes microfibrils of cellulose that surround all plant cells. Cellulose is made of sugar (β,1‐4 glucan) and accessing the sugar in cellulose for biofuels is of critical importance to stem the use of fossil fuels and avoid competition with food crops and pristine lands associated with starch‐based biofuel production. The recalcitrance of cellulose to enzymatic conversion to a fermentable form of sugar is related to the degree of hydrogen bonding or crystallization of the glucan chain. Herein, we isolate the first viable low biomass‐crystallinity mutant by screening for altered cell wall structure using X‐ray scattering as well as screening for enzymatic conversion efficiency on a range of cell wall mutants in the model plant Arabidopsis thaliana (L.) Heynh. Through detailed analysis of the kinetics of bioconversion we identified a mutant that met both selection criteria. This mutant is ixr1‐2, which contains a mutation in a highly conserved consensus sequence among the C‐terminal transmembrane regions within CESA3. A 34% lower biomass crystallization index and 151% improvement in the efficiency of conversion from raw biomass to fermentable sugars was measured relative to that of wild type (Col‐0). Recognizing the inherent ambiguities with an insoluble complex substrate like cellulose and how little is still understood regarding the regulation of CESA we propose a general model for how to manipulate CESA enzymes to improve the recalcitrance of cellulose to enzymatic hydrolysis. This study also raises intriguing possibilities as to the functional importance of transmembrane anchoring in CESA complex and microfibril formation.     

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.     

Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana

The cell wall determines the shape of plant cells and is also the primary interface for pathogen interactions. The structure of the cell wall can be modified in response to developmental and environmental cues, for example to strengthen the wall and to create barriers to pathogen ingress. The ectopic lignin 1-1 and 1-2 (eli1-1 and eli1-2) mutations lead to an aberrant deposition of lignin, a complex phenylpropanoid polymer. We show that the eli1 mutants occur in the cellulose synthase gene CESA3 in Arabidopsis thaliana and cause reduced cellulose synthesis, providing further evidence for the function of multiple CESA subunits in cellulose synthesis. We show that reduced levels of cellulose synthesis, caused by mutations in cellulose synthase genes and in genes affecting cell expansion, activate lignin synthesis and defense responses through jasmonate and ethylene and other signaling pathways. These observations suggest that mechanisms monitoring cell wall integrity can activate lignification and defense responses.     

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.     

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.     

KORRIGAN1 Interacts Specifically with Integral Components of the Cellulose Synthase Machinery

Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane–cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.     

Spatio‐temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo‐vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP‐labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co‐localisation studies using GFP–CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast‐associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP–CESA from doughnut‐shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP–CESA density diminished, whereas GFP–CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP–CESA in clathrin‐containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose‐deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.     

Some aspects of cellulose biosynthesis

The paper is focused on two groups of proteins inevitably important for cellulose biosynthesis in vascular plants. These are cellulose synthases and chitinase-like proteins. Cellulose synthases have been the subject of much research, and current conceptions and recent findings are reviewed in this paper. Severe effects of mutations and expression analysis have recently shown that chitinase-like proteins are crucial components of cellulose biosynthesis. However, understanding of their precise function is missed. Further research is to be prompted by an effective idea on it. I propose that chitinase-like proteins could play a role in the assembly of nascent glucan chains into microfibrills. Therefore, cellulose synthases and chitinase-like proteins are possibly sequential elements of the cellulose biosynthesis.