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.     

POM-POM2/cellulose synthase interacting1 is essential for the functional association of cellulose synthase and microtubules in Arabidopsis

In plants, regulation of cellulose synthesis is fundamental for morphogenesis and plant growth. Cellulose is synthesized at the plasma membrane, and the orientation of synthesis is guided by cortical microtubules; however, the guiding mechanism is currently unknown. We show that the conditional root elongation pom2 mutants are impaired in cell elongation, fertility, and microtubule-related functions. Map-based cloning of the POM-POM2 locus revealed that it is allelic to CELLULOSE SYNTHASE INTERACTING1 (CSI1). Fluorescently tagged POM2/CSI1s associated with both plasma membrane-located cellulose synthases (CESAs) and post-Golgi CESA-containing compartments. Interestingly, while CESA insertions coincided with cortical microtubules in the pom2/csi1 mutants, the microtubule-defined movement of the CESAs was significantly reduced in the mutant. We propose that POM2/CSI1 provides a scaffold between the CESAs and cortical microtubules that guide cellulose synthesis.     

Making parallel lines meet: Transferring information from microtubules to extracellular matrix

The extracellular matrix is constructed beyond the plasma membrane, challenging mechanisms for its control by the cell. In plants, the cell wall is highly ordered, with cellulose microfibrils aligned coherently over a scale spanning hundreds of cells. To a considerable extent, deploying aligned microfibrils determines mechanical properties of the cell wall, including strength and compliance. Cellulose microfibrils have long been seen to be aligned in parallel with an array of microtubules in the cell cortex. How do these cortical microtubules affect the cellulose synthase complex? This question has stood for as many years as the parallelism between the elements has been observed, but now an answer is emerging. Here, we review recent work establishing that the link between microtubules and microfibrils is mediated by a protein named cellulose synthase-interacting protein 1 (CSI1). The protein binds both microtubules and components of the cellulose synthase complex. In the absence of CSI1, microfibrils are synthesized but their alignment becomes uncoupled from the microtubules, an effect that is phenocopied in the wild type by depolymerizing the microtubules. The characterization of CSI1 significantly enhances knowledge of how cellulose is aligned, a process that serves as a paradigmatic example of how cells dictate the construction of their extracellular environment.     

Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis

The shape of plants depends on cellulose, a biopolymer that self-assembles into crystalline, inextensible microfibrils (CMFs) upon synthesis at the plasma membrane by multi-enzyme cellulose synthase complexes (CSCs). CSCs are displaced in directions predicted by underlying parallel arrays of cortical microtubules, but CMFs remain transverse in cells that have lost the ability to expand unidirectionally as a result of disrupted microtubules. These conflicting findings suggest that microtubules are important for some physico-chemical property of cellulose that maintains wall integrity. Using X-ray diffraction, we demonstrate that abundant microtubules enable a decrease in the degree of wall crystallinity during rapid growth at high temperatures. Reduced microtubule polymer mass in the mor1-1 mutant at high temperatures is associated with failure of crystallinity to decrease and a loss of unidirectional expansion. Promotion of microtubule bundling by over-expressing the RIC1 microtubule-associated protein reduced the degree of crystallinity. Using live-cell imaging, we detected an increase in the proportion of CSCs that track in microtubule-free domains in mor1-1, and an increase in the CSC velocity. These results suggest that microtubule domains affect glucan chain crystallization during unidirectional cell expansion. Microtubule disruption had no obvious effect on the orientation of CMFs in dark-grown hypocotyl cells. CMFs at the outer face of the hypocotyl epidermal cells had highly variable orientation, in contrast to the transverse CMFs on the radial and inner periclinal walls. This suggests that the outer epidermal mechanical properties are relatively isotropic, and that axial expansion is largely dependent on the inner tissue layers.     

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.     

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 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.     

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.     

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     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule–cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

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.     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule-cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

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.     

Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification,localization, and function in cellulose biosynthesis and osmotic stress responses

Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation of KOR1 at tonoplasts. The degree of successful TGN localization of KOR1 agreed well with in vivo-complementation efficacy of the rsw2–1 mutant, suggesting non-catalytic functions in the TGN. A dynamic interaction network involving microtubules, CSCs, KOR1, and currently unidentified glycoprotein component(s) likely determines stress-triggered re-organization of cellulose biosynthesis and resumption of cell-wall growth under stress.     

CELLULOSE SYNTHASE INTERACTIVE3 Regulates Cellulose Biosynthesis in Both a Microtubule-Dependent and Microtubule-Independent Manner in Arabidopsis

Anisotropic plant cell growth depends on the coordination between the orientation of cortical microtubules and the orientation of nascent cellulose microfibrils. CELLULOSE SYNTHASE INTERACTIVE1 (CSI1) is a key scaffold protein that guides primary cellulose synthase complexes (CSCs) along cortical microtubules during cellulose biosynthesis. Here, we investigated the function of the CSI1-like protein, CSI3, in Arabidopsis thaliana. Similar to CSI1, CSI3 associates with primary CSCs in vitro, colocalizes with CSCs in vivo, and exhibits the same plasma membrane localization and bidirectional motility as CSI1. However, ProCSI1:GFP-CSI3 cannot complement the anisotropic cell growth defect in csi1 mutants, suggesting that CSI3 is not functionally equivalent to CSI1. Also, the colocalization ratio between CSI1 and CSI3 is low, which may suggest heterogeneity within the CSC population. csi1 csi3 double mutants showed an enhanced cell expansion defect as well as an additive reduction of CSC velocities, and CSI3 dynamics are dependent on CSI1 function. We propose that CSI3 is an important regulator of plant cellulose biosynthesis and plant anisotropic cell growth that modulates the velocity of CSCs in both a microtubule-dependent and microtubule-independent manner.