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.     

Temperature-dependent changes in hydrogen bonds in cellulose Ialpha studied by infrared spectroscopy in combination with perturbation-correlation moving-window two-dimensional correlation spectroscopy: comparison with cellulose Ibeta

Our recent IR study demonstrated that hydrogen-bond structure in cellulose Ibeta drastically changes around 220 degrees C (Watanabe et al. Biomacromolecules 2006, 7, 3164). In the present study, temperature-dependent IR spectra of cellulose Ialpha from 30 to 260 degrees C were analyzed by use of perturbation-correlation moving-window two-dimensional correlation spectroscopy. It was observed that as in the case of cellulose Ibeta abrupt changes in the hydrogen-bond structure occur around 220 degrees C in cellulose Ialpha. It was also revealed that although weakly hydrogen-bonded OH groups in Ibeta are stable below 230 degrees C thermal oxidation of those in Ialpha is accelerated around 220 degrees C. In this way, the present study has clarified a difference between the thermal behavior of Ialpha and that of Ibeta at the functional group level. Our result suggests that the drastic change in the hydrogen-bond structure around 220 degrees C makes cellulose Ialpha much more unstable than Ibeta.     

Interconversion of the Ialpha and Ibeta crystalline forms of cellulose by bending

Bending cellulose in a plane normal to the hydrogen-bonded sheets of chains causes a longitudinal displacement of the sheets with respect to one another. The magnitude of this displacement is shown to be sufficient to interconvert the Ialpha and Ibeta forms of cellulose within a bending angle of 39 degrees when the curvature of the sheets of chains comprising the microfibril is modelled as a series of concentric circular arcs. Bending through an angle of 90 degrees is more than sufficient to convert the Ialpha form into Ibeta and back again. Cellulose microfibrils emerging from the cellulose synthase complex in the plasma membrane must bend sharply before they can lie parallel with the inner face of the cell wall. The scale of the changes induced by bending is sufficient to ensure that whatever crystal form would be expected from the geometry of the biosynthetic complex, it is likely be radically altered before the cellulose is incorporated into the cell wall.     

Characterization of the crystalline structure of cellulose using static and dynamic FT-IR spectroscopy

The cellulose structure is a factor of major importance for the strength properties of wood pulp fibers. The ability to characterize small differences in the crystalline structures of cellulose from fibers of different origins is thus highly important. In this work, dynamic FT-IR spectroscopy has been further explored as a method sensitive to cellulose structure variations. Using a model system of two different celluloses, the relation between spectral information and the relative cellulose Ialpha content was investigated. This relation was then used to determine the relative cellulose Ialpha content in different pulps. The estimated cellulose I allomorph compositions were found to be reasonable for both unbleached and bleached chemical pulps. In addition, it was found that the dynamic FT-IR spectroscopy technique had the potential to indicate possible correlation field splitting peaks of cellulose Ibeta.     

Cellulose synthesis in two secondary cell wall processes in a single cell type

Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cell''s function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process.     

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

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.     

Cyclic AMP-binding proteins and protamine kinases in porcine thyroid cytosol.

Partial purification of cyclic AMP-binding proteins from porcine thyroid cytosol was performed by gel filtration on Bio Gel 1.5 m followed by ion exchange chromatography on DEAE Sephadex A25. Three fractions presenting cyclic AMP-binding activities were resolved by gel filtration (I, II, III). Approximate molecular weights were respectively 280 000, 145 000 and 65 000. Fraction I was further resolved into two peaks (Ialpha and Ibeta) on DEAE-Sephadex A25. Fractions I, Ialpha, Ibeta comigrated with protein kinase activity whereas peaks II and III did not. These fractions differed with respect to the folling characteristics: rate and stability of cyclic AMP binding to isolated fractions were differently affected by pH (4.0 or 7.5). Electrophoretic mobility on polyacrylamide gels (5%) of fractions preincubated with cyclic [3H]AMP showed similar mobilities for Ialpha, Ibeta or II (Rf 0.37) whereas fraction III displayed a much greater mobility (RF 0.73); Scatchard plots were linear for fractions Ialpha, II and III with an apparent Kd in the same range (2 to 5 nM) whereas fraction Ibeta generated a biphasic plot with Kd 0.4 nM and 20 nM; cyclic [3H] AMP added to fraction I, Ialpha or Ibeta generated a cyclic [3H] AMP-binding protein complex of lower molecular weight as shown by Sephadex G 150 filtration; on the basis of the elution volume, this complex was not distinguished from fraction II. In the course of this work, we separated at the first step of purification (Bio Gel 1.5 m) a protein kinase not associated with cyclic AMP binding activity which exhibited marked specificity for protamine as compared to histone II A.     

Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes

Plant cell walls are degraded by glycoside hydrolases that often contain noncatalytic carbohydrate-binding modules (CBMs), which potentiate degradation. There are currently 11 sequence-based cellulose-directed CBM families; however, the biological significance of the structural diversity displayed by these protein modules is uncertain. Here we interrogate the capacity of eight cellulose-binding CBMs to bind to cell walls. These modules target crystalline cellulose (type A) and are located in families 1, 2a, 3a, and 10 (CBM1, CBM2a, CBM3a, and CBM10, respectively); internal regions of amorphous cellulose (type B; CBM4-1, CBM17, CBM28); and the ends of cellulose chains (type C; CBM9-2). Type A CBMs bound particularly effectively to secondary cell walls, although they also recognized primary cell walls. Type A CBM2a and CBM10, derived from the same enzyme, displayed differential binding to cell walls depending upon cell type, tissue, and taxon of origin. Type B CBMs and the type C CBM displayed much weaker binding to cell walls than type A CBMs. CBM17 bound more extensively to cell walls than CBM4-1, even though these type B modules display similar binding to amorphous cellulose in vitro. The thickened primary cell walls of celery collenchyma showed significant binding by some type B modules, indicating that in these walls the cellulose chains do not form highly ordered crystalline structures. Pectate lyase treatment of sections resulted in an increased binding of cellulose-directed CBMs, demonstrating that decloaking cellulose microfibrils of pectic polymers can increase CBM access. The differential recognition of cell walls of diverse origin provides a biological rationale for the diversity of cellulose-directed CBMs that occur in cell wall hydrolases and conversely reveals the variety of cellulose microstructures in primary and secondary cell walls.     

Electron diffraction from the primary wall of cotton fibers

Summary Electron diffraction patterns have been obtained from selected areas of disencrusted microfibrils isolated from the primary cell wall of cotton fibers. The resultant fiber diagram has the same meridional repeat distance as a corresponding pattern of secondary wall microfibrils but differs markedly in the equatorial reflections. The primary wall diagram displays only two strong equatorial reflections centered at 0.570 nm and 0.416 nm. The similarity of these spacings with those of cellulose IV suggests that the crystalline structure of the primary wall cellulose is similar to that of cellulose IV_I and is best explained in term of native cellulose I crystals having good longitudinal coherence (i.e., coherence along the length of the microfibrils) but with poor lateral organization of the network of inter chain hydrogen bonds. Similar results were also obtained for other primary wall specimens.     

Compatibility properties of R483, a member of the I plasmid complex.

R483, an I pilus-determining plasmid previously reported as belonging to a distinct incompatibility group Ibeta, proved to be an atypical Ialpha plasmid; in a growing culture, the degree of inhibition of replication of one Ialpha plasmid by the presence of another was not uniform within the Ialpha group.     

A priori crystal structure prediction of native celluloses

The packing of beta-1,4-glucopyranose chains has been modeled to further elaborate the molecular structures of native cellulose microfibrils. A chain pairing procedure was implemented that evaluates the optimal interchain distance and energy for all possible settings of the two chains. Starting with a rigid model of an isolated chain, its interaction with a second chain was studied at various helix-axis translations and mutual rotational orientations while keeping the chains at van der Waals separation. For each setting, the sum of the van der Waals and hydrogen-bonding energy was calculated. No energy minimization was performed during the initial screening, but the energy and interchain distances were mapped to a three-dimensional grid, with evaluation of parallel settings of the cellulose chains. The emergence of several energy minima suggests that parallel chains of cellulose can be paired in a variety of stable orientations. A further analysis considered all possible parallel arrangements occurring between a cellulose chain pair and a further cellulose chain. Among all the low-energy three-chain models, only a few of them yield closely packed three-dimensional arrangements. From these, unit-cell dimensions as well as lattice symmetry were derived; interestingly two of them correspond closely to the observed allomorphs of crystalline native cellulose. The most favorable structural models were then optimized using a minicrystal procedure in conjunction with the MM3 force field. The two best crystal lattice predictions were for a triclinic (P(1)) and a monoclinic (P2(1)) arrangement with unit cell dimensions a = 0.63, b = 0.69, c = 1.036 nm, alpha = 113.0, beta = 121.1, gamma = 76.0 degrees, and a = 0.87, b = 0.75, c = 1.036 nm, gamma = 94.1 degrees, respectively. They correspond closely to the respective lattice symmetry and unit-cell dimensions that have been reported for cellulose Ialpha and cellulose Ibeta allomorphs. The suitability of the modeling protocol is endorsed by the agreement between the predicted and experimental unit-cell dimensions. The results provide pertinent information toward the construction of macromolecular models of microfibrils.     

Identification of a conserved residue responsible for the autoinhibition of cGMP-dependent protein kinase Ialpha and beta

We isolated a constitutively active form of cGMP-dependent protein kinase Ialpha (cGK Ialpha) by PCR-driven random mutagenesis. The replacement of Ile-63 by Thr in the autoinhibitory domain results in the enhancement of autophosphorylation and the basal kinase activity in the absence of cGMP. The hydrophobicity at position 63 is essential for the inactive state of cGK Ialpha, and Ile-78 of cGK Ibeta is also required for the autoinhibitory property. Furthermore, cGK Ialpha (Ile-63-Thr) is constitutively active in vivo. These findings suggest that a conserved residue in the autoinhibitory domain was involved in the autoinhibition of both cGK Is.     

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.     

Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana

In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall.     

Expression of a mutant form of cellulose synthase AtCesA7 causes dominant negative effect on cellulose biosynthesis

Cellulose synthase catalytic subunits (CesAs) have been implicated in catalyzing the biosynthesis of cellulose, the major component of plant cell walls. Interactions between CesA subunits are thought to be required for normal cellulose synthesis, which suggests that incorporation of defective CesA subunits into cellulose synthase complex could potentially cause a dominant effect on cellulose synthesis. However, all CesA mutants so far reported have been shown to be recessive in terms of cellulose synthesis. In the course of studying the molecular mechanisms regulating secondary wall formation in fibers, we have found that a mutant allele of AtCesA7 gene in the fra5 (fragile fiber 5) mutant causes a semidominant phenotype in the reduction of fiber cell wall thickness and cellulose content. The fra5 missense mutation occurred in a conserved amino acid located in the second cytoplasmic domain of AtCesA7. Overexpression of the fra5 mutant cDNA in wild-type plants not only reduced secondary wall thickness and cellulose content but also decreased primary wall thickness and cell elongation. In contrast, overexpression of the fra6 mutant form of AtCesA8 did not cause any reduction in cell wall thickness and cellulose content. These results suggest that the fra5 mutant protein may interfere with the function of endogenous wild-type CesA proteins, thus resulting in a dominant negative effect on cellulose biosynthesis.