Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation

Cellulose synthase complexes (CSCs) play a central role in plant cell wall formation. Their dynamic behavior at the plasma membrane leads to the deposition of cellulose microfibrils into the apoplastic space, thereby shaping the architecture and mechanical properties of the cell wall. Although previous imaging studies have provided important insights into CSC dynamics and localization, standardized and reproducible workflows for quantitative measurements of CSC speed and density remain limited. Here, we present a reproducible live-cell imaging and analysis workflow for quantifying the speed and density of fluorescently labeled CSCs at the plasma membrane in Arabidopsis thaliana. The protocol integrates optimized spinning-disk confocal imaging, surface-based projection of z-stack recordings, automated detection of diffraction-limited CSCs foci, and kymograph-based speed measurements using freely available tools in Fiji. While selected steps, such as region of interest definition and parameter selection for spot detection or trajectory analysis, remain user-guided, these decisions are constrained to well-defined stages within an otherwise standardized pipeline, thereby reducing variability and improving reproducibility across experiments. The workflow has been validated across multiple tissues, reporter lines, genetic backgrounds, and perturbation conditions in Arabidopsis and enables robust comparative analysis of CSC dynamics. Beyond CSCs, this workflow is expected to be adaptable to other fluorescently labeled proteins that appear as diffraction-limited foci at or near the plasma membrane.Key features• Enables accurate CSC speed and density measurements during both primary and secondary cell wall formation using spinning-disk confocal time-lapse imaging.• Combines surface-projection, kymograph analysis, and high-throughput particle detection to quantify CSC dynamics even in crowded or low-signal plasma membrane regions.• Provides a standardized analysis workflow validated across multiple Arabidopsis genotypes, including inducible systems and mutant backgrounds that possess altered cell wall biosynthesis.• Applicable to any fluorescently labeled diffraction-limited foci at or near the plasma membrane, extending the workflow beyond CSCs.

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 (irx1cesa8, irx3cesa7 and irx5cesa4). 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 irx1cesa8 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 biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.

Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating CESA4Sha and CESA4Bay. In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of CESA4Bay, CESA4Sha, and the reference genome CESA4Col. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the CESA4Sha promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element.

Primary cell walls are deposited and remodeled during cell division and expansion. Secondary cell walls are deposited in specialized cells after the expansion phase. It is presently unknown whether and how these processes are interrelated. The Arabidopsis (Arabidopsis thaliana) MUR10 gene is required for normal primary cell wall carbohydrate composition in mature leaves as well as for normal plant growth, hypocotyl strength, and fertility. The overall sugar composition of young mur10 seedlings is not significantly altered; however, the relative proportion of pectin side chains is shifted toward an increase in 1 --> 5-alpha-arabinan relative to 1 --> 4-beta-galactan. mur10 seedlings display reduced fucogalactosylation of tightly cell wall-bound xyloglucan. Expression levels of genes encoding either nucleotide sugar interconversion enzymes or glycosyl transferases, known to be involved in primary and secondary cell wall biosynthesis, are generally unaffected; however, the CesA7 transcript is specifically suppressed in the mur10-1 allele. The MUR10 locus is identical with the CesA7 gene, which encodes a cellulose catalytic subunit previously thought to be specifically involved in secondary cell wall formation. The xylem vessels in young mur10 hypocotyls are collapsed and their birefringence is lost. Moreover, a fucogalactosylated xyloglucan epitope is reduced and a 1 --> 5-alpha-arabinan epitope increased in every cell type in mur10 hypocotyls, including cells that do not deposit secondary walls. mur10 also displays altered distribution of an arabinogalactan-protein epitope previously associated with xylem differentiation and secondary wall thickening. This work indicates the existence of a mechanism that senses secondary cell wall integrity and controls biosynthesis or structural remodeling of primary cell walls and cellular differentiation.

Plant cell walls contain a meshwork of cellulose fibers embedded into a matrix of other carbohydrate and non-carbohydrate-based biopolymers. This composite material exhibits extraordinary properties, from stretchable and pliable cell boundaries to solid protective shells. Cellulose, a linear glucose polymer, is synthesized and secreted across the plasma membrane by cellulose synthase (CesA), of which plants express multiple isoforms. Different subsets of CesA isoforms are necessary for primary and secondary cell wall biogenesis. Here, we structurally and functionally characterize the Glycine max (soybean) primary cell wall CesAs CesA1, CesA3, and CesA6. The CesA isoforms exhibit robust in vitro catalytic activity. Cryo-electron microscopy analyses reveal their assembly into homotrimeric complexes in vitro in which each CesA protomer forms a cellulose-conducting transmembrane channel with a large lateral opening. Biochemical and co-purification analyses demonstrate that different CesA isoforms interact in vitro, leading to synergistic cellulose biosynthesis. Interactions between CesA trimers are only observed between different CesA isoforms and require the class-specific region (CSR). The CSR forms a hook-shaped extension of CesA’s catalytic domain at the cytosolic water-lipid interface. Negative stain and cryo-electron microscopy analyses of mixtures of different CesA isoform trimers reveal their side-by-side arrangement into loose clusters. Our data suggest a model by which CesA homotrimers of different isoforms assemble into cellulose synthase complexes to synthesize and secrete multiple cellulose chains for microfibril formation. Inter-trimer interactions are mediated by fuzzy interactions between their CSR extension.

In higher plants, cellulose is synthesized by plasma membrane-localized cellulose synthase complexes (CSCs). Arabidopsis thaliana GH9A1/KORRIGAN1 is a membrane-bound, family 9 glycosyl hydrolase that is important for cellulose synthesis in both primary and secondary cell walls. Most previously identified korrigan1 mutants show severe phenotypes such as embryo lethality; therefore, the role of GH9A1 in cellulose synthesis remains unclear. Here, we report a novel A577V missense mutation, designated jiaoyao1 (jia1), in the second of the glycosyl hydrolase family 9 active site signature motifs in GH9A1. jia1 is defective in cell expansion in dark-grown hypocotyls, roots, and adult plants. Consistent with its defect in cell expansion, this mutation in GH9A1 resulted in reduced cellulose content and reduced CSC velocity at the plasma membrane. Green fluorescent protein-GH9A1 is associated with CSCs at multiple locations, including the plasma membrane, Golgi, trans-Golgi network, and small CESA-containing compartments or microtubule-associated cellulose synthase compartments, indicating a tight association between GH9A1 and CSCs. GH9A1A577V abolishes the endoglucanase activity of GH9A1 in vitro but does not affect its interaction with CESAs in vitro, suggesting that endoglucanase activity is important for cellulose synthesis. Interestingly, jia1 results in both cellulose microfibril and microtubule disorganization. Our study establishes the important role of endoglucanase in cellulose synthesis and cellulose microfibril organization in plants.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar

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.

BackgroundNAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored.ResultsWe overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area.ConclusionsThis study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan endo-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of XTH4 and XTH9 abundantly expressed in cambial regions during secondary growth of Arabidopsis (Arabidopsis thaliana). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither XTH4 nor XTH9 was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including THESEUS1 and WALL ASSOCIATED KINASE2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.

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 physiological effects of an experimental herbicide and cellulose synthesis inhibitor, N2-(1-ethyl-3-phenylpropyl)-6-(1-fluoro-1-methylethyl)-1,3,5-triazine-2,4-diamine, called AE F150944, are described. In the aminotriazine molecular class, AE F150944 is structurally distinct from other known cellulose synthesis inhibitors. It specifically inhibits crystalline cellulose synthesis in plants without affecting other processes that were tested. The effects of AE F150944 on dicotyledonous plants were tested on cultured mesophyll cells of Zinnia elegans L. cv. Envy, which can be selectively induced to expand via primary wall synthesis or to differentiate into tracheary elements via secondary wall synthesis. The IC50 values during primary and secondary wall synthesis in Z. elegans were 3.91 x 10(-8) M and 3.67 x 10(-9) M, respectively. The IC50 in suspension cultures of the monocot Sorghum halapense (L.) Pers., which were dividing and synthesizing primary walls, was 1.67 x 10(-10) M. At maximally inhibitory concentrations, 18-33% residual crystalline cellulose synthesis activity remained, with the most residual activity observed during primary wall synthesis in Z. elegans. Addition to Z. elegans cells of two other cellulose synthesis inhibitors, 1 microM 2,6-dichlorobenzonitrile and isoxaben, along with AE F150944 did not eliminate the residual cellulose synthesis, indicating little synergy between the three inhibitors. In differentiating tracheary elements, AE F150944 inhibited the deposition of detectable cellulose into patterned secondary wall thickenings, which was correlated with delocalization of lignin as described previously for 2, 6-dichlorobenzonitrile. Freeze-fracture electron microscopy showed that the plasma membrane below the patterned thickenings of AE F150944-treated tracheary elements was depleted of cellulose-synthase-containing rosettes, which appeared to be inserted intact into the plasma membrane followed by their rapid disaggregation. AE F150944 also inhibited cellulose-dependent growth in the rosette-containing alga, Spirogyra pratensis, but it did not inhibit cellulose synthesis in Acetobacter xylinum or Dictyostelium discoideum, both of which synthesize cellulose via linear terminal complexes. Therefore, AE F150944 may inhibit crystalline cellulose synthesis by destabilizing plasma membrane rosettes.

Plant cells are surrounded by a carbohydrate-rich extracellular matrix known as the cell wall. Primary cell walls are laid down around dividing and elongating cells and consist largely of the polysaccharides cellulose, hemicelluloses, and pectin along with approximately 10% protein. Specific cells such as xylem vessels and fibers lay down a secondary wall rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin. Most of the models depict the plant cell wall as a matrix of separate polysaccharides. However, the recent identification of a proteoglycan that contains covalently attached pectin and xylan indicates that at least some of these wall glycans exist as domains within a single glycopolymer and that current models of the wall need to be revised. Furthermore, several cell wall biosynthesis mutants, including the secondary cell wall mutant irregular xylem (irx) 8, are affected in multiple cell wall polymers making it challenging to define the biochemical function of the mutated gene. The goal of this review is to provide a background for studying genes which encode secondary cell wall biosynthetic proteins whose mutation affects multiple wall polymers including xylan and lignin. We first review the phenotypes of the irx mutants and then summarize the current understanding of the structure and synthesis of xylan and lignin along with a review of transcription factors known to affect secondary wall synthesis. This review is intended to serve as a resource for those studying genes that encode proteins involved in the synthesis of plant secondary wall lignin and xylan.

The secondary cell wall in plant cells is a highly organized structure that provides mechanical support to the tissue and the entire plant body. Glycine soja, the presumed ancestor of domesticated soybean, differs from the domesticated soybean in an array of morphological characters. The remarkable difference is the stem phenotype, which is often attributed to the thickness of secondary cell walls. Hence, it is of great importance to understand the genetic basis underlying the regulation of secondary wall thickening in these two species. Here, we report that GlycineNST1A/B are functional orthologs of AtNST1/2, which have been shown to act as the master switches of secondary cell wall thickening. GlycineNST1A/B are preferentially expressed in organs undergoing secondary wall deposition, and GmNST1A/B are expressed at twofold the level of GsNST1A/B in mature stem. The overexpression of GsNST1A/B disrupts overall plant development by inducing ectopic deposition of massive secondary walls in normally parenchyma cells in Arabidopsis. Furthermore, expression analyses suggest that the ectopic secondary wall is achieved by upregulating the expression of a series of cell wall‐associated transcription factors and secondary wall biosynthetic genes. Together, these results indicate that GsNST1A/B, as the key regulators, are capable of activating the entire secondary wall biosynthetic pathway in Arabidopsis, and also suggest that the genetic circuit of secondary wall development is conserved between Fabaceae and Brassiaceae.