Primary Wall Cellulose Research Articles

Tip growth defective1 interacts with cellulose synthase A3 to regulate cellulose biosynthesis in Arabidopsis.

AtTIP1 physically and genetically interacts with AtCESA3. AtCESA3 undergoes S-acylation, possibly mediated by AtTIP1, suggesting a specific role of AtTIP1 in cellulose biosynthesis and plant development. S-acylation is a reversible post-translational lipid modification of proteins catalyzed by protein S-acyl transferases (PATs). S-acylation is important for various biological molecular mechanisms including cellulose biosynthesis. Cellulose is synthesized by the cellulose synthase A (CESA) complexes (CSCs) at the plasma membrane. However, specific PAT involving in cellulose biosynthesis has not been identified and the precise mechanism by which PAT regulates the CESAs is largely unknown. Here, we report isolation of tip1-5, an allele of Tip Growth Defective1 (AtTIP1/AtPAT24) with a premature stop codon. tip1-5 genetically interacts with ixr1-2, a point mutant of AtCESA3 which encodes a catalytic subunit of CSC synthesizing primary wall cellulose. We show that AtTIP1 physically interacts with AtCESA3. AtCESA3 undergoes S-acylation, which is possibly mediated by AtTIP1, suggesting a functional relationship between AtTIP1 and AtCESA3. Moreover, the interfascicular fiber cells in the primary inflorescence stems of tip1-5 ixr1-2 double mutant contain thinner cell walls and significantly less crystalline cellulose compared to the single mutants. These results highlight the positive regulation of AtTIP1 in cellulose biosynthesis, and a specific role of AtPAT in plant development.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

Cellulose Structural Polymorphism in Plant Primary Cell Walls Investigated by High-Field 2D Solid-State NMR Spectroscopy and Density Functional Theory Calculations.

The native cellulose of bacterial, algal, and animal origins has been well studied structurally using X-ray and neutron diffraction and solid-state NMR spectroscopy, and is known to consist of varying proportions of two allomorphs, Iα and Iβ, which differ in hydrogen bonding, chain packing, and local conformation. In comparison, cellulose structure in plant primary cell walls is much less understood because plant cellulose has lower crystallinity and extensive interactions with matrix polysaccharides. Here we have combined two-dimensional magic-angle-spinning (MAS) solid-state nuclear magnetic resonance (solid-state NMR) spectroscopy at high magnetic fields with density functional theory (DFT) calculations to obtain detailed information about the structural polymorphism and spatial distributions of plant primary-wall cellulose. 2D (13)C-(13)C correlation spectra of uniformly (13)C-labeled cell walls of several model plants resolved seven sets of cellulose chemical shifts. Among these, five sets (denoted a-e) belong to cellulose in the interior of the microfibril while two sets (f and g) can be assigned to surface cellulose. Importantly, most of the interior cellulose (13)C chemical shifts differ significantly from the (13)C chemical shifts of the Iα and Iβ allomorphs, indicating that plant primary-wall cellulose has different conformations, packing, and hydrogen bonding from celluloses of other organisms. 2D (13)C-(13)C correlation experiments with long mixing times and with water polarization transfer revealed the spatial distributions and matrix-polysaccharide interactions of these cellulose structures. Celluloses f and g are well mixed chains on the microfibril surface, celluloses a and b are interior chains that are in molecular contact with the surface chains, while cellulose c resides in the core of the microfibril, outside spin diffusion contact with the surface. Interestingly, cellulose d, whose chemical shifts differ most significantly from those of bacterial, algal, and animal cellulose, interacts with hemicellulose, is poorly hydrated, and is targeted by the protein expansin during wall loosening. To obtain information about the C6 hydroxymethyl conformation of these plant celluloses, we carried out DFT calculations of (13)C chemical shifts, using the Iα and Iβ crystal structures as templates and varying the C5-C6 torsion angle. Comparison with the experimental chemical shifts suggests that all interior cellulose favor the tg conformation, but cellulose d also has a similar propensity to adopt the gt conformation. These results indicate that cellulose in plant primary cell walls, due to their interactions with matrix polysaccharides, and has polymorphic structures that are not a simple superposition of the Iα and Iβ allomorphs, thus distinguishing them from bacterial and animal celluloses.

Prediction of the structures of the plant-specific regions of vascular plant cellulose synthases and correlated functional analysis

Seed plants express cellulose synthase (CESA) protein isoforms with non-redundant functions, but how the isoforms function differently is unknown. Compared to bacterial cellulose synthases, CESAs have two insertions in the large cytosolic loop: the relatively well-conserved Plant Conserved Region (P-CR) and a Class Specific Region (CSR) that varies between CESAs. Absent any atomic structure of a plant CESA, we used ab initio protein structure prediction and molecular modeling to explore how these plant-specific regions may modulate CESA function. We modeled P-CR and CSR peptides from Arabidopsis thaliana CESAs representing the six clades of seed plant CESAs. As expected, the predicted wild type P-CR structures were similar. Modeling of the mutant P-CR of Atcesa8 R362K (fra6) suggested that changes in local structural stability and surface electrostatics may cause the mutant phenotype. Among CSRs within CESAs required for primary wall cellulose synthesis, the amino sequence and the modeled arrangement of helices was most similar in AtCESA1 and AtCESA3. Genetic complementation of known Arabidopsis mutants showed that the CSRs of AtCESA1 and AtCESA3 can function interchangeably in vivo. Analysis of protein surface electrostatics led to ideas about how the surface charges on CSRs may mediate protein–protein interactions. Refined modeling of the P-CR and CSR regions of GhCESA1 from cotton modified their tertiary structures, spatial relationships to the catalytic domain, and preliminary predictions about CESA oligomer formation. Cumulatively, the results provide structural clues about the function of plant-specific regions of CESA.

Solid-state NMR investigations of cellulose structure and interactions with matrix polysaccharides in plant primary cell walls.

Until recently, the 3D architecture of plant cell walls was poorly understood due to the lack of high-resolution techniques for characterizing the molecular structure, dynamics, and intermolecular interactions of the wall polysaccharides in these insoluble biomolecular mixtures. We introduced multidimensional solid-state NMR (SSNMR) spectroscopy, coupled with (13)C labelling of whole plants, to determine the spatial arrangements of macromolecules in near-native plant cell walls. Here we review key evidence from 2D and 3D correlation NMR spectra that show relatively few cellulose-hemicellulose cross peaks but many cellulose-pectin cross peaks, indicating that cellulose microfibrils are not extensively coated by hemicellulose and all three major polysaccharides exist in a single network rather than two separate networks as previously proposed. The number of glucan chains in the primary-wall cellulose microfibrils has been under active debate recently. We show detailed analysis of quantitative (13)C SSNMR spectra of cellulose in various wild-type (WT) and mutant Arabidopsis and Brachypodium primary cell walls, which consistently indicate that primary-wall cellulose microfibrils contain at least 24 glucan chains.

An integrated genomic and metabolomic framework for cell wall biology in rice.

BackgroundPlant cell walls are complex structures that full-fill many diverse functions during plant growth and development. It is therefore not surprising that thousands of gene products are involved in cell wall synthesis and maintenance. However, functional association for the majority of these gene products remains obscure. One useful approach to infer biological associations is via transcriptional coordination, or co-expression of genes. This approach has proved useful for several biological processes. Nevertheless, combining co-expression with other large-scale measurements may improve the biological inferences.ResultsIn this study, we used a combined approach of co-expression and cell wall metabolomics to obtain new insight into cell wall synthesis in rice. We initially created a weighted gene co-expression network from publicly available datasets, and then established a comprehensive cell wall dataset by determining cell wall compositions from 29 tissues that almost cover the whole life cycle of rice. We subsequently combined the datasets through the conversion of co-expressed gene modules into eigen-vectors, representing expression profiles for the genes in the modules, and performed comparative analyses against the cell wall contents. Here, we made three major discoveries. First, we confirmed our approach by finding primary and secondary wall cellulose biosynthesis modules, respectively. Second, we found co-expressed modules that strongly correlated with re-organization of the secondary cell walls and with modifications and degradation of hemicellulosic structures. Third, we inferred that at least one module is likely to play a regulatory role in the production of G-rich lignification.ConclusionsHere, we integrated transcriptomic associations and cell wall metabolism and found that certain co-expressed gene modules are positively correlated with distinct cell wall characteristics. We propose that combining multiple data-types, such as coordinated transcription and cell wall analyses, may be a useful approach to glean new insight into biological processes. The combination of multiple datasets, as illustrated here, can further improve the functional inferences that typically are generated via a single type of datasets. In addition, our data extend the typical co-expression approach to allow deeper insight into cell wall biology in rice.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-596) contains supplementary material, which is available to authorized users.

TRICHOME BIREFRINGENCEand Its HomologAT5G01360Encode Plant-Specific DUF231 Proteins Required for Cellulose Biosynthesis in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) trichome birefringence (tbr) mutant has severely reduced crystalline cellulose in trichomes, but the molecular nature of TBR was unknown. We determined TBR to belong to the plant-specific DUF231 domain gene family comprising 46 members of unknown function in Arabidopsis. The genes harbor another plant-specific domain, called the TBL domain, which contains a conserved GDSL motif known from some esterases/lipases. TBR and TBR-like3 (TBL3) are transcriptionally coordinated with primary and secondary CELLULOSE SYNTHASE (CESA) genes, respectively. The tbr and tbl3 mutants hold lower levels of crystalline cellulose and have altered pectin composition in trichomes and stems, respectively, tissues generally thought to contain mainly secondary wall crystalline cellulose. In contrast, primary wall cellulose levels remain unchanged in both mutants as measured in etiolated tbr and tbl3 hypocotyls, while the amount of esterified pectins is reduced and pectin methylesterase activity is increased in this tissue. Furthermore, etiolated tbr hypocotyls have reduced length with swollen epidermal cells, a phenotype characteristic for primary cesa mutants or the wild type treated with cellulose synthesis inhibitors. Taken together, we show that two TBL genes contribute to the synthesis and deposition of secondary wall cellulose, presumably by influencing the esterification state of pectic polymers.

Features of the primary wall CESA complex in wild type and cellulose-deficient mutants of Arabidopsis thaliana

Evidence from genetics, co-precipitation and bimolecular fluorescence complementation suggest that three CESAs implicated in making primary wall cellulose in Arabidopsis thaliana form a complex. This study shows the complex has a Mr of approximately 840 kDa in detergent extracts and that it has undergone distinctive changes when extracts are prepared from some cellulose-deficient mutants. The mobility of CESAs 1, 3, and 6 in a Triton-soluble microsomal fraction subject to blue native polyacrylamide gel electrophoresis was consistent with a Mr of about 840 kDa. An antibody specific to any one CESA pulled down all three CESAs consistent with their occupying the same 840 kDa complex. In rsw1, a CESA1 missense mutant, extracts of seedlings grown at the permissive temperature have an apparently normal CESA complex that was missing from extracts of seedlings grown at the restrictive temperature where CESAs precipitated independently. In prc1-19, with no CESA6, CESAs 1 and 3 were part of a 420 kDa complex in extracts of light-grown seedlings that was absent from extracts of dark-grown seedlings where the CESAs precipitated independently. Two CESA3 missense mutants retained apparently normal CESA complexes as did four cellulose-deficient mutants defective in proteins other than CESAs. The 840 kDa complex could contain six CESA subunits and, since loss of plasma membrane rosettes accompanies its loss in rsw1, the complex could form one of the six particles which electron microscopy reveals in rosettes.

Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana

In all land plants, cellulose is synthesized from hexameric plasma membrane complexes. Indirect evidence suggests that in vascular plants the complexes involved in primary wall synthesis contain three distinct cellulose synthase catalytic subunits (CESAs). In this study, we show that CESA3 and CESA6 fused to GFP are expressed in the same cells and at the same time in the hypocotyl of etiolated seedlings and migrate with comparable velocities along linear trajectories at the cell surface. We also show that CESA3 and CESA6 can be coimmunoprecipitated from detergent-solubilized extracts, their protein levels decrease in mutants for either CESA3, CESA6, or CESA1 and CESA3, CESA6 and also CESA1 can physically interact in vivo as shown by bimolecular fluorescence complementation. We also demonstrate that CESA6-related CESA5 and CESA2 are partially, but not completely, redundant with CESA6 and most likely compete with CESA6 for the same position in the cellulose synthesis complex. Using promoter-beta-glucuronidase fusions we show that CESA5, CESA6, and CESA2 have distinct overlapping expression patterns in hypocotyl and root corresponding to different stages of cellular development. Together, these data provide evidence for the existence of binding sites for three distinct CESA subunits in primary wall cellulose synthase complexes, with two positions being invariably occupied by CESA1 and CESA3, whereas at least three isoforms compete for the third position. Participation of the latter three isoforms might fine-tune the CESA complexes for the deposition of microfibrils at distinct cellular growth stages.

Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis.

In higher plants, cellulose is synthesized at the plasma membrane by the cellulose synthase (CESA) complex. The catalytic core of the complex is believed to be composed of three types of CESA subunits. Indirect evidence suggests that the complex associated with primary wall cellulose deposition consists of CESA1, -3, and -6 in Arabidopsis thaliana. However, phenotypes associated with mutations in two of these genes, CESA1 and -6, suggest unequal contribution by the different CESAs to overall enzymatic activity of the complex. We present evidence that the primary complex requires three unique types of components, CESA1-, CESA3-, and CESA6-related, for activity. Removal of any of these components results in gametophytic lethality due to pollen defects, demonstrating that primary-wall cellulose synthesis is necessary for pollen development. We also show that the CESA6-related CESAs are partially functionally redundant.

Hydration effects on spacing of primary-wall cellulose microfibrils: a small angle X-ray scattering study

Celery collenchyma cell walls are typical of primary plant cell walls in their composition but contain unusually well-oriented cellulose microfibrils that are packed with more regularity than normal, permitting small-angle X-ray scattering (SAXS) experiments that would not otherwise be possible. Small-angle scattering data were obtained for the cell walls in essentially their native state and for isolated cellulose, in a fibrous form that retained the physical shape and microfibril orientation of the native cell walls. The scattering patterns showed a distinct peak attributed to the interference contribution to the convolution of form and interference functions. The position of the peak attributed to the interference function implied a mean centre-to-centre microfibril spacing of approximately 3.2 nm in dry isolated cellulose and 3.8 nm in dry cell walls. Hydration increased the mean microfibril spacing in the cell walls to 5.4 nm but had only a small effect on the mean microfibril spacing of isolated cellulose. In the scattering profile from intact, hydrated cell walls it was just possible to discern the position of the first Bessel minimum, from which a microfibril diameter in the range 3.1–3.6 nm may be estimated. This estimate is likely to include attached hemicellulose chains. Porod plots of scattering intensity indicated a relatively sharp interface between microfibrils and their immediate surroundings. The SAXS data imply that cellulose microfibrils 2.6–3.0 nm in diameter are not quite in lateral contact with one another in the isolated cellulose and are augmented by hemicelluloses and separated by readily hydrated matrix polysaccharides in the native plant cell wall.

Microfibril diameter in celery collenchyma cellulose: X-ray scattering and NMR evidence

Cellulose isolated from celery collenchyma is typical of the low-crystallinity celluloses that can be isolated from primary cell-walls of higher plants, except that it is oriented with high uniformity. The diameter of the microfibrils of celery collenchyma cellulose was estimated by three separate approaches: 13C NMR measurement of the ratio of surface to interior chains; estimation of the dimensions of the crystalline lattice from wide angle X-ray scattering (WAXS) measurements using the Scherrer equation; and the observation that microfibrils of this form of cellulose have the unusual property of packing into an irregular array from which small angle X-ray scattering (SAXS) shows features of both form and interference functions. The interference function contributing to the SAXS pattern implied a mean microfibril centre-to-centre distance of 3.6 nm, providing an upper limit for the diameter. However modelling of the scattering pattern from an irregular array of microfibrils showed that the observed scattering curve could be matched at a range of diameters down to 2.4 nm, with the intervening space more or less sparsely occupied by hemicellulose chains. The lateral extent of the crystalline lattice normal to the 200 plane was estimated as a minimum of 2.4 nm by WAXS through the Scherrer equation, and a diameter of 2.6 nm was implied by the surface: volume ratio determined by 13C NMR. The WAXS and NMR measurements both depended on the assumption that the surface chains were positioned within an extension of the crystalline lattice. The reliability of this assumption is uncertain. If the surface chains deviated from the lattice, both the WAXS and the NMR data would imply larger microfibril diameters within the range consistent with the SAXS pattern. The evidence presented is therefore all consistent with microfibril diameters from about 2.4 to 3.6 nm, larger than has previously been suggested for primary-wall cellulose. Some degree of aggregation may have occurred during the isolation of the cellulose, but the larger microfibril diameters within the range proposed are a consequence of the novel interpretation of the experimental data from WAXS and NMR and are consistent with previously published data if these are similarly interpreted.

Chimeric Proteins Suggest That the Catalytic and/or C-Terminal Domains Give CesA1 and CesA3 Access to Their Specific Sites in the Cellulose Synthase of Primary Walls

CesA1 and CesA3 are thought to occupy noninterchangeable sites in the cellulose synthase making primary wall cellulose in Arabidopsis (Arabidopsis thaliana L. Heynh). With domain swaps and deletions, we show that sites C terminal to transmembrane domain 2 give CesAs access to their individual sites and, from dominance and recessive behavior, deduce that certain CesA alleles exclude others from accessing each site. Constructs that swapped or deleted N-terminal domains were stably transformed into the wild type and into the temperature-sensitive mutants rsw1 (Ala-549Val in CesA1) and rsw5 (Pro-1056Ser in CesA3). Dominant-positive behavior was assayed as root elongation at the restrictive temperature and dominant-negative effects were observed at the permissive temperature. A protein with the catalytic and C-terminal domains of CesA1 and the N-terminal domain of CesA3 promoted growth only in rsw1 consistent with it accessing the CesA1 site even though it contained the CesA3 N-terminal domain. A protein having the CesA3 catalytic and C-terminal domains linked to the CesA1 N-terminal domain dramatically affected growth, but only in the CesA3 mutant. This is consistent with the operation of the same access rule taking this chimeric protein to the CesA3 site. In this case, however, the transgene behaved as a genotype-specific dominant negative, causing a 60% death rate in rsw5, but giving no visible phenotype in wild type or rsw1. We therefore hypothesize that possession of CesA3(WT) protects Columbia and rsw1 from the lethal effects of this chimeric protein, whereas the mutant protein (CesA3(rsw5)) does not.

Variation of xyloglucan substitution pattern affects the sorption on celluloses with different degrees of crystallinity

The sorption of xyloglucan (XG) on cellulose is a basic feature of the supramolecular assembly of plant cell walls. The binding to cellulose of xyloglucan fractions from Rubus fruticosus suspension-cultured cells with different substitution patterns was assayed on celluloses having various degrees of crystallinity between 20 and 95%. The primary structure of XGs differing in their Xyl/Glc ratio affected their binding to cellulose. The less substituted XGs gave the highest binding yields. Selective removal of the terminal fucosyl residues of XGs differentially affected the binding depending on the crystallinity of cellulose. The results showed large variations on the way cellulose crystallinity affects the binding interaction of XGs. Interestingly, one of the highest binding capacities was exhibited by the primary cell wall cellulose isolated from the actual R. fruticosus cells which also had the lowest crystallinity. Differences in binding to primary wall cellulose appeared to be inversely related to the global substitution of the glucan main chain of XGs.

Structural details of crystalline cellulose from higher plants.

It is commonly assumed that cellulose from higher plants contains the Ialpha and Ibeta crystalline allomorphs together with surface and disordered chains. For cellulose Ialpha, the evidence for its presence in higher plants is restricted to the C-4 signals in the solid-state (13)C NMR spectrum, which match those of crystalline cellulose Ialpha from algal sources. Algal cellulose Ialpha can be converted to the Ibeta form by high-temperature annealing. We used this approach to generate cellulose samples differing in Ibeta content from flax fibers and celery collenchyma, which respectively are representative of textile (secondary-wall) and primary-wall cellulose. It was then possible to isolate the detailed spectral contributions of the surface, Ibeta and Ialpha-like phases from linear combinations of the observed (13)C NMR and FTIR spectra. The (13)C NMR spectra resembled those of highly crystalline tunicate or algal cellulose Ibeta and Ialpha, with slight differences implying increased disorder and minor conformational discrepancies. The FTIR spectrum of the Ibeta form was closely similar to its more crystalline counterparts, but the FTIR spectrum of the Ialpha form was not. In addition to increased bandwith indicative of lower order, it showed substantial differences in the profile of hydroxyl stretching bands. These results confirm that higher plants synthesize cellulose Ibeta but show that the Ialpha-like chains, although conformationally quite similar to crystalline algal cellulose Ialpha, sit in a different hydrogen-bonding environment in higher plants. The differences are presumably occasioned by the small diameter of the crystallites and the influence of the crystallite surface on chain packing.

Cellulose biosynthesis in plants: from genes to rosettes.

Modern techniques of gene cloning have identified the CesA genes as encoding the probable catalytic subunits of the plant CelS, the cellulose synthase enzyme complex visualized in the plasma membrane as rosettes. At least 10 CesA isoforms exist in Arabidopsis and have been shown by mutant analyses to play distinct role/s in the cellulose synthesis process. Functional specialization within this family includes differences in gene expression, regulation and, possibly, catalytic function. Current data points towards some CesA isoforms potentially being responsible for initiation or elongation of the recently identified sterol beta-glucoside primer within different cell types, e.g. those undergoing either primary or secondary wall cellulose synthesis. Different CesA isoforms may also play distinct roles within the rosette, and there is some circumstantial evidence that CesA genes may encode the catalytic subunit of the mixed linkage glucan synthase or callose synthase. Various other proteins such as the Korrigan endocellulase, sucrose synthase, cytoskeletal components, Rac13, redox proteins and a lipid transfer protein have been implicated to be involved in synthesizing cellulose but, apart from CesAs, only Korrigan has been definitively linked with cellulose synthesis. These proteins should prove valuable in identifying additional CelS components.

Functional analysis of the cellulose synthase genes CesA1, CesA2, and CesA3 in Arabidopsis.

Polysaccharide analyses of mutants link several of the glycosyltransferases encoded by the 10 CesA genes of Arabidopsis to cellulose synthesis. Features of those mutant phenotypes point to particular genes depositing cellulose predominantly in either primary or secondary walls. We used transformation with antisense constructs to investigate the functions of CesA2 (AthA) and CesA3 (AthB), genes for which reduced synthesis mutants are not yet available. Plants expressing antisense CesA1 (RSW1) provided a comparison with a gene whose mutant phenotype (Rsw1(-)) points mainly to a primary wall role. The antisense phenotypes of CesA1 and CesA3 were closely similar and correlated with reduced expression of the target gene. Reductions in cell length rather than cell number underlay the shorter bolts and stamen filaments. Surprisingly, seedling roots were unaffected in both CesA1 and CesA3 antisense plants. In keeping with the mild phenotype compared with Rsw1(-), reductions in total cellulose levels in antisense CesA1 and CesA3 plants were at the borderline of significance. We conclude that CesA3, like CesA1, is required for deposition of primary wall cellulose. To test whether there were important functional differences between the two, we overexpressed CesA3 in rsw1 but were unable to complement that mutant's defect in CesA1. The function of CesA2 was less obvious, but, consistent with a role in primary wall deposition, the rate of stem elongation was reduced in antisense plants growing rapidly at 31 degrees C.

Mercerization of primary wall cellulose and its implication for the conversion of cellulose I→cellulose II

The mercerization of homogenized primary wall cellulose extracted fromsugar beet pulp was investigated by transmission electron microscopy (TEM),X-ray diffraction together with 13C CP-MAS NMR, and FT-IR spectroscopy.For samples resulting from acid extraction, mercerization began at 9% NaOH, whereasfor samples purified by alkaline treatment, the mercerization started at 10%NaOH. The change in morphology when going from cellulose I to cellulose II wasspectacular, as all the microfibrillar cellulose morphology disappeared duringthe treatment. This change in morphology was very drastic as soon as the NaOHconcentrations were increased beyond 8 and 9% for the acid and alkalinepreparedsamples, respectively. On the other hand, the conversion was found to be moreprogressive in terms of increasing NaOH concentration when the transformationwas analyzed by X-ray diffraction or spectroscopy. Our observations of themercerization of isolated cellulose microfibrils are consistent with theconceptof cellulose microfibrils made of parallel chains in cellulose I and crystalsofcellulose II consisting of antiparallel chains.

Quantitative analysis for the cellulose I α crystalline phase in developing wood cell walls

FT-IR and X-ray analyses were employed to determine the relative ratio of cellulose I α and I β crystalline phases present in each developmental stage of coniferous tracheid cell wall formation. The IR spectra showed that initially the I α phase occupies 50% of the crystalline regions in the primary cell wall cellulose and this value drops to 20% after ceasing of the cell enlarging growth for the formation of the secondary wall cellulose (the remaining regions are composed of the I β phase). Although it is reasonable that the content for I β , which is stress-reduced crystalline form, was higher in the secondary wall formation (Kataoka Y, and Kondo T. Macromolecules 1996;29:6356–6358) it is more interesting that during the crystallization of stress-induced I α cellulose for the primary wall the stress-reduced I β is also possible to be crystallized in an alternative way. This means that throughout the period the I α -causing stress may not be necessarily kept loaded. In light of our previously reported hypothesis (Kataoka Y. and Kondo T. Macromolecules 1998;31:760–764) for the formation of I α phase due to cellular growing stresses in the primary wall cellulose, such an alternating on–off stress effect to account for the occurrence of both I α and I β phases might be related to a biological growth system in coniferous wood cells.

FT-IR Microscopic Analysis of Changing Cellulose Crystalline Structure during Wood Cell Wall Formation

A Fourier transform infrared (FT-IR) spectrometer equipped with a microscopic accessory was used to monitor the cellulose crystalline structure at each developmental stage of coniferous tracheid cell wall formation. The spectra showed that the cellulose in the primary cell wall was rich in the metastable Iα crystalline form and interestingly had a higher crystallinity than the secondary wall cellulose composed mainly of the stable Iβ crystalline phase. These results indicate the presence of an in vivo physical force at the cell surface which may stress the primary wall cellulose during the crystallization. Moreover, the primary wall cellulose was oriented parallel to the enlarging direction of growing cells. Thus, we consider that the cellular growing stress generated between the plasma membrane and the primary wall may elongate just-biosynthesized β-glucan chains and cause them to crystallize with a higher crystallinity of the metastable Iα phase, as is the case for crystallization occurring during the d...