Orientation Of Microfibrils Research Articles

Morphological and structural characterization of the attachment system in aerial roots of Syngonium podophyllum.

The attachment of aerial roots of Syngonium podophyllum involves a multi-step process adjusted by multi-scale structures. Helical-crack root hairs are first found in the attachment system, representing specialized structures for surface anchorage. The morphological variability of attachment organs reflects diverse climbing strategies. One such anchoring mode in clinging-climbers involves the time-dependent interaction between roots and the support: By naturally occurring adhesive roots with root hairs, the plant can ascend on supports of any shape and size. As a typical root-climber, Syngonium podophyllum develops elongate aerial roots at nodes. Here, we studied its attachment behavior from the external morphology to the internal structure in detail. Through SEM and LM observation on several root-substrate interfaces, we suggested that the attachment of aerial roots was mediated by a multi-step process, in which root hairs played significant roles in releasing mucilaginous substance and securing the durable anchorage. We summarized all the types of shape changes of root hairs with particular focus on the abnormal transition from a tube to a helical-crack ribbon. We demonstrated our understanding with respect to the formation of the helical-crack root hairs, based on the structural evidence of cellulose microfibrils orientation on the cell wall lamellae. The helical-crack root hairs serving as energy-dissipating units retard the failure of adhesion under high winds and loads.

Helicoidal Organization of Chitin in the Cuticle of the Migratory Locust Requires the Function of the Chitin Deacetylase2 Enzyme (LmCDA2)

In the three-dimensional extracellular matrix of the insect cuticle, horizontally aligned microfibrils composed of the polysaccharide chitin and associated proteins are stacked either parallel to each other or helicoidally. The underlying molecular mechanisms that implement differential chitin organization are largely unknown. To learn more about cuticle organization, we sought to study the role of chitin deacetylases (CDA) in this process. In the body cuticle of nymphs of the migratory locust Locusta migratoria, helicoidal chitin organization is changed to an organization with unidirectional microfibril orientation when LmCDA2 expression is knocked down by RNA interference. In addition, the LmCDA2-deficient cuticle is less compact suggesting that LmCDA2 is needed for chitin packaging. Animals with reduced LmCDA2 activity die at molting, underlining that correct chitin organization is essential for survival. Interestingly, we find that LmCDA2 localizes only to the initially produced chitin microfibrils that constitute the apical site of the chitin stack. Based on our data, we hypothesize that LmCDA2-mediated chitin deacetylation at the beginning of chitin production is a decisive reaction that triggers helicoidal arrangement of subsequently assembled chitin-protein microfibrils.

The Impact of Microfibril Orientations on the Biomechanics of Plant Cell Walls and Tissues.

The microscopic structure and anisotropy of plant cell walls greatly influence the mechanical properties, morphogenesis, and growth of plant cells and tissues. The microscopic structure and properties of cell walls are determined by the orientation and mechanical properties of the cellulose microfibrils and the mechanical properties of the cell wall matrix. Viewing the shape of a plant cell as a square prism with the axis aligning with the primary direction of expansion and growth, the orientation of the microfibrils within the side walls, i.e. the parts of the cell walls on the sides of the cells, is known. However, not much is known about their orientation at the upper and lower ends of the cell. Here we investigate the impact of the orientation of cellulose microfibrils within the upper and lower parts of the plant cell walls by solving the equations of linear elasticity numerically. Three different scenarios for the orientation of the microfibrils are considered. We also distinguish between the microstructure in the side walls given by microfibrils perpendicular to the main direction of the expansion and the situation where the microfibrils are rotated through the wall thickness. The macroscopic elastic properties of the cell wall are obtained using homogenization theory from the microscopic description of the elastic properties of the cell wall microfibrils and wall matrix. It is found that the orientation of the microfibrils in the upper and lower parts of the cell walls affects the expansion of the cell in the lateral directions and is particularly important in the case of forces acting on plant cell walls and tissues.

Stomatal Function Requires Pectin De-methyl-esterification of the Guard Cell Wall

SummaryStomatal opening and closure depends on changes in turgor pressure acting within guard cells to alter cell shape [1]. The extent of these shape changes is limited by the mechanical properties of the cells, which will be largely dependent on the structure of the cell walls. Although it has long been observed that guard cells are anisotropic due to differential thickening and the orientation of cellulose microfibrils [2], our understanding of the composition of the cell wall that allows them to undergo repeated swelling and deflation remains surprisingly poor. Here, we show that the walls of guard cells are rich in un-esterified pectins. We identify a pectin methylesterase gene, PME6, which is highly expressed in guard cells and required for stomatal function. pme6-1 mutant guard cells have walls enriched in methyl-esterified pectin and show a decreased dynamic range in response to triggers of stomatal opening/closure, including elevated osmoticum, suggesting that abrogation of stomatal function reflects a mechanical change in the guard cell wall. Altered stomatal function leads to increased conductance and evaporative cooling, as well as decreased plant growth. The growth defect of the pme6-1 mutant is rescued by maintaining the plants in elevated CO2, substantiating gas exchange analyses, indicating that the mutant stomata can bestow an improved assimilation rate. Restoration of PME6 rescues guard cell wall pectin methyl-esterification status, stomatal function, and plant growth. Our results establish a link between gene expression in guard cells and their cell wall properties, with a corresponding effect on stomatal function and plant physiology.

Cell wall matrix polysaccharide distribution and cortical microtubule organization: two factors controlling mesophyll cell morphogenesis in land plants.

This work investigates the involvement of local differentiation of cell wall matrix polysaccharides and the role of microtubules in the morphogenesis of mesophyll cells (MCs) of three types (lobed, branched and palisade) in the dicotyledon Vigna sinensis and the fern Asplenium nidus. Homogalacturonan (HGA) epitopes recognized by the 2F4, JIM5 and JIM7 antibodies and callose were immunolocalized in hand-made leaf sections. Callose was also stained with aniline blue. We studied microtubule organization by tubulin immunofluorescence and transmission electron microscopy. In both plants, the matrix cell wall polysaccharide distribution underwent definite changes during MC differentiation. Callose constantly defined the sites of MC contacts. The 2F4 HGA epitope in V. sinensis first appeared in MC contacts but gradually moved towards the cell wall regions facing the intercellular spaces, while in A. nidus it was initially localized at the cell walls delimiting the intercellular spaces, but finally shifted to MC contacts. In V. sinensis, the JIM5 and JIM7 HGA epitopes initially marked the cell walls delimiting the intercellular spaces and gradually shifted in MC contacts, while in A. nidus they constantly enriched MC contacts. In all MC types examined, the cortical microtubules played a crucial role in their morphogenesis. In particular, in palisade MCs, cortical microtubule helices, by controlling cellulose microfibril orientation, forced these MCs to acquire a truncated cone-like shape. Unexpectedly in V. sinensis, the differentiation of colchicine-affected MCs deviated completely, since they developed a cell wall ingrowth labyrinth, becoming transfer-like cells. The results of this work and previous studies on Zea mays (Giannoutsou et al., Annals of Botany 2013; 112: : 1067-1081) revealed highly controlled local cell wall matrix differentiation in MCs of species belonging to different plant groups. This, in coordination with microtubule-dependent cellulose microfibril alignment, spatially controlled cell wall expansion, allowing MCs to acquire their particular shape.

Microtubule encounter-based catastrophe in Arabidopsis cortical microtubule arrays.

BackgroundThe cortical microtubules (CMTs) that line the plasma membrane of interphase plant cells are extensively studied owing to their importance in forming cell walls, and their usefulness as a model system for the study of MT dynamic instability and acentrosomal MT organization. CMTs influence the orientation and structure of cellulose microfibrils in the cell wall by cooperatively forming arrays of varied patterns from parallel to netted. These CMT patterns are controlled by the combined activities of MT dynamic instability and MT-MT interactions. However, it is an open question as to how CMT patterns may feedback to influence CMT dynamics and interactions.ResultsTo address this question, we investigated the effects of CMT array patterning on encounter-based CMT catastrophe, which occurs when one CMT grows into another and is unable to cross over. We hypothesized that the varied CMT angles present in disordered (mixed CMTs) arrays will create more opportunities for MT-MT interactions, and thus increase encounter-based catastrophe rates and distribution. Using live-cell imaging of Arabidopsis cotyledon and leaf epidermal cells, we found that roughly 87 % of catastrophes occur via the encounter-based mechanism, with the remainder occurring without encounter (free). When comparing ordered (parallel) and disordered (mixed orientation) CMT arrays, we found that disordered configurations show higher proportions of encounter-based catastrophe relative to free. Similarly, disordered CMT arrays have more catastrophes in general than ordered arrays. Encounter-based catastrophes were associated with frequent and sustained periods of pause prior to depolymerization, and CMTs with tight anchoring to the plasma membrane were more prone to undergo encounter-based catastrophe than weakly-attached ones. This suggests that encounter-based catastrophe has a mechanical basis, wherein MTs form physical barriers to one another. Lastly, we show that the commonly used measure of catastrophe frequencies (Fcat) can also be influenced by CMT ordering and plasma membrane anchoring.ConclusionsOur observations add a new layer of complexity to our current understanding of MT organization in plants, showing that not only do individual CMT dynamics influence CMT array organization, but that CMT organization itself has a strong effect on the behavior of individual MTs.

Mechanism of dynamic reorientation of cortical microtubules due to mechanical stress

Directional growth caused by gravitropism and corresponding bending of plant cells has been explored since 19th century, however, many aspects of mechanisms underlying the perception of gravity at the molecular level are still not well known. Perception of gravity in root and shoot gravitropisms is usually attributed to gravisensitive cells, called statocytes, which exploit sedimentation of macroscopic and heavy organelles, amyloplasts, to sense the direction of gravity. Gravity stimulus is then transduced into distal elongation zone, which is several mm far from statocytes, where it causes stretching. It is suggested that gravity stimulus is conveyed by gradients in auxin flux. We propose a theoretical model that may explain how concentration gradients and/or stretching may indirectly affect the global orientation of cortical microtubules, attached to the cell membrane and induce their dynamic reorientation perpendicular to the gradients. In turn, oriented microtubule arrays direct the growth and orientation of cellulose microfibrils, forming part of the cell external skeleton and determine the shape of the cell. Reorientation of microtubules is also observed in reaction to light in phototropism and mechanical bending, thus suggesting universality of the proposed mechanism.

Non-invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy.

Cellulose microfibrils represent the major scaffold of plant cell walls. Different packing and orientation of the microfibrils at the microscopic scale determines the macroscopic properties of cell walls and thus affect their functions with a profound effect on plant survival. We developed a polarized Raman microspectroscopic method to determine cellulose microfibril orientation within rice plant cell walls. Employing an array of point measurements as well as area imaging and subsequent Matlab-assisted data processing, we were able to characterize the distribution of cellulose microfibril orientation in terms of director angle and anisotropy magnitude. Using this approach we detected differences between wild type rice plants and the rice brittle culm mutant, which shows a more disordered cellulose microfibril arrangement, and differences between different tissues of a wild type rice plant. This novel non-invasive Raman imaging approach allows for quantitative assessment of cellulose fiber orientation in cell walls of herbaceous plants, an important advancement in cell wall characterization.

Constitutive modeling of a paper fiber in cyclic loading applications

The tensile response of dense fiber-based materials like paper or paperboard is mainly dependent of the properties of the fibers, which store most of the elastic energy. In this paper, we investigate the influence of geometrical and material parameters on the mechanical response of the pulp fibers used in paper manufacturing. We developed a three-dimensional finite element model of the fiber, which accounts for microfibril orientation of cellulose fibril, and the presence of lignin in the secondary cell wall. The results showed that the change in the microfibril orientation upon axial straining is mainly a geometrical effect, and is independent of the material properties of the fiber, as long as the deformations are elastic. Plastic strain accelerates the change in microfibril orientation and thus makes it material-dependent. The results also showed that the elastic modulus of the fiber has a non-linear dependency on microfibril angle, with elastic modulus being more sensitive to the change of microfibril angle around small initial values of microfibril angles. Based on numerical results acquired from a 3D fiber model supported by available experimental evidence, we propose an anisotropic-kinematic hardening plasticity model for a fiber within a beam framework. The proposed fiber model is capable of reproducing the main features of the cyclic tensile response of a pulp fiber, such as stiffening due to changing microfibril angle. The constitutive model of the fiber was implemented in a finite-element model of the fiber network. By using the fiber network model, we estimated the level of strain that fiber segments accumulate before the typical failure strain of the entire network is reached.

Structural characterization of a mixed-linkage glucan deficient mutant reveals alteration in cellulose microfibril orientation in rice coleoptile mesophyll cell walls.

The CELLULOSE SYNTHASE-LIKE F6 (CslF6) gene was previously shown to mediate the biosynthesis of mixed-linkage glucan (MLG), a cell wall polysaccharide that is hypothesized to be tightly associated with cellulose and also have a role in cell expansion in the primary cell wall of young seedlings in grass species. We have recently shown that loss-of-function cslf6 rice mutants do not accumulate MLG in most vegetative tissues. Despite the absence of a structurally important polymer, MLG, these mutants are unexpectedly viable and only show a moderate growth compromise compared to wild type. Therefore these mutants are ideal biological systems to test the current grass cell wall model. In order to gain a better understanding of the role of MLG in the primary wall, we performed in-depth compositional and structural analyses of the cell walls of 3 day-old rice seedlings using various biochemical and novel microspectroscopic approaches. We found that cellulose content as well as matrix polysaccharide composition was not significantly altered in the MLG deficient mutant. However, we observed a significant change in cellulose microfibril bundle organization in mesophyll cell walls of the cslf6 mutant. Using synchrotron source Fourier Transform Mid-Infrared (FTM-IR) Spectromicroscopy for high-resolution imaging, we determined that the bonds associated with cellulose and arabinoxylan, another major component of the primary cell walls of grasses, were in a lower energy configuration compared to wild type, suggesting a slightly weaker primary wall in MLG deficient mesophyll cells. Taken together, these results suggest that MLG may influence cellulose deposition in mesophyll cell walls without significantly affecting anisotropic growth thus challenging MLG importance in cell wall expansion.

Tubulin perturbation leads to unexpected cell wall modifications and affects stomatal behaviour in Populus.

Cortical microtubules are integral to plant morphogenesis, cell wall synthesis, and stomatal behaviour, presumably by governing cellulose microfibril orientation. Genetic manipulation of tubulins often leads to abnormal plant development, making it difficult to probe additional roles of cortical microtubules in cell wall biogenesis. Here, it is shown that expressing post-translational C-terminal modification mimics of α-tubulin altered cell wall characteristics and guard cell dynamics in transgenic Populus tremula x alba that otherwise appear normal. 35S promoter-driven transgene expression was high in leaves but unusually low in xylem, suggesting high levels of tubulin transgene expression were not tolerated in wood-forming tissues during regeneration of transformants. Cellulose, hemicellulose, and lignin contents were unaffected in transgenic wood, but expression of cell wall-modifying enzymes, and extractability of lignin-bound pectin and xylan polysaccharides were increased in developing xylem. The results suggest that pectin and xylan polysaccharides deposited early during cell wall biogenesis are more sensitive to subtle tubulin perturbation than cellulose and matrix polysaccharides deposited later. Tubulin perturbation also affected guard cell behaviour, delaying drought-induced stomatal closure as well as light-induced stomatal opening in leaves. Pectins have been shown to confer cell wall flexibility critical for reversible stomatal movement, and results presented here are consistent with microtubule involvement in this process. Taken together, the data show the value of growth-compatible tubulin perturbations for discerning microtubule functions, and add to the growing body of evidence for microtubule involvement in non-cellulosic polysaccharide assembly during cell wall biogenesis.

Detection and distribution of cell growth regulators and cellulose microfibrils during the development of Lopesia sp. galls on Lonchocarpus cultratus (Fabaceae)

Gall-inducing insects cause their plant hosts’ cells to redifferentiate, divide, and elongate, which are processes mediated by cell growth regulators. In this study, the histochemical detection of these regulators, and of cellulose microfibrils were performed in a gall induced by Lopesia sp. (Cecidomyiidae) on Lonchocarpus cultratus (Vell.) A.M.G.Azevedo & H.C.Lima. The co-occurrence of reactive oxygen species, (poly)phenols, flavonoids, and indole-3-acetic acid are age-independent and are detected at the sites of cell hypertrophy. Nevertheless, the intensity of the reactions may vary from young to mature galls. First, changes in the axis of cell elongation are observed from the non-galled leaflets to the young galls, in the cortical parenchyma and in the nutritive tissue. During maturation, the elongation axis of the nutritive cells changes again. In young galls, sites of hyperplasia and hypertrophy co-occur, and in mature galls, the orientation of cellulose microfibrils determines the predominant anisotropic pattern of cell expansion. The detection of (poly)phenols and indole-3-acetic acid, reactive oxygen species, and flavonoids coincide with the sites of cell elongation and division. These results denote a chemical balance between the regulation of growth and the avoidance of cell death at gall sites. The rearrangement of cellulose microfibrils coordinates the anisotropic expansion, which determines the development of the tissue projections both to adaxial and abaxial leaflet lamina, typical to this gall morphotype.

The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl

Complex shapes in biology depend on the ability of cells to shift from isotropic to anisotropic growth during development. In plants, this growth symmetry breaking reflects changes in the extensibility of the cell walls. The textbook view is that the direction of turgor-driven cell expansion depends on the cortical microtubule (CMT)-mediated orientation of cellulose microfibrils. Here, we show that this view is incomplete at best. We used atomic force microscopy (AFM) to study changes in cell-wall mechanics associated with growth symmetry breaking within the hypocotyl epidermis. We show that, first, growth symmetry breaking is preceded by an asymmetric loosening of longitudinal, as compared to transverse, anticlinal walls, in the absence of a change in CMT orientation. Second, this wall loosening is triggered by the selective de-methylesterification of cell-wall pectin in longitudinal walls, and, third, the resultant mechanical asymmetry is required for the growth symmetry breaking. Indeed, preventing or promoting pectin de-methylesterification, respectively, increased or decreased the stiffness of all the cell walls, but in both cases reduced the growth anisotropy. Finally, we show that the subsequent CMT reorientation contributes to the consolidation of the growth axis but is not required for the growth symmetry breaking. We conclude that growth symmetry breaking is controlled at a cellular scale by bipolar pectin de-methylesterification, rather than by the cellulose-dependent mechanical anisotropy of the cell walls themselves. Such a cell asymmetry-driven mechanism is comparable to that underlying tip growth in plants but also anisotropic cell growth in animal cells.

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.

The Delignification Pattern of Ailanthus excelsa Wood by Inonotus hispidus (Bull.: Fr.) P. Karst.

In vitro laboratory decay tests on Ailanthus excelsa Roxb. wood revealed that I. hispidus exhibits a combination of both white-rot and soft-rot patterns of wood decay. Early stages of wood decay showed dissolution of the middle lamella as well as defibration and localized delignification of fiber walls; vessels, axial, and ray parenchyma remained unaltered. Delignification commenced from the middle lamellae at the cell corners without any marked effect on the primary and secondary wall layers. In later stages of growth, the species produces typical soft-rot decay pattern by forming erosion channels through the S2 layers of fiber walls, transverse bore holes in the cell walls, and erosion channels alongside/following the orientation of cellulose microfibrils. The rays showed signs of cell wall alterations only after the extensive damage to the fiber walls. After 120 days of incubation, the vessels also showed localized delignification, the erosion of pits, and separation from associated xylem elements. The extensive weight losses under natural and in vitro decayed wood as well as the very soft nature of severely degraded wood indicate that I. hispidus alters wood strength and stiffness.

Cellulose structure and lignin distribution in normal and compression wood of the Maidenhair tree (Ginkgo biloba L.).

We studied in detail the mean microfibril angle and the width of cellulose crystals from the pith to the bark of a 15-year-old Maidenhair tree (Ginkgo biloba L.). The orientation of cellulose microfibrils with respect to the cell axis and the width and length of cellulose crystallites were determined using X-ray diffraction. Raman microscopy was used to compare the lignin distribution in the cell wall of normal/opposite and compression wood, which was found near the pith. Ginkgo biloba showed a relatively large mean microfibril angle, varying between 19° and 39° in the S2 layer, and the average width of cellulose crystallites was 3.1-3.2 nm. Mild compression wood without any intercellular spaces or helical cavities was observed near the pith. Slit-like bordered pit openings and a heavily lignified S2L layer confirmed the presence of compression wood. Ginkgo biloba showed typical features present in the juvenile wood of conifers. The microfibril angle remained large over the 14 annual rings. The entire stem disc, with a diameter of 18 cm, was considered to consist of juvenile wood. The properties of juvenile and compression wood as well as the cellulose orientation and crystalline width indicate that the wood formation of G. biloba is similar to that of modern conifers.

OsKinesin-13A is an active microtubule depolymerase involved in glume length regulation via affecting cell elongation.

Grain size is an important trait influencing both the yield and quality of rice and its major determinant is glume size. However, how glume size is regulated remains largely unknown. Here, we report the characterization of OsKinesin-13A, which regulates cell elongation and glume length in rice. The mutant of OsKinesin-13A, sar1, displayed length reduction in grains and other organs including internodes, leaves and roots. The grain phenotype in sar1 was directly caused by reduction in glume length, which in turn restricted caryopsis size. Histological results revealed that length decrease in sar1 organs resulted from abnormalities in cell elongation. The orientation of cellulose microfibrils was defective in sar1. Consistently, sar1 showed reduced transverse orientation of cortical microtubules. Further observations demonstrated that microtubule turnover was decreased in sar1. OsKinesin-13A was shown to be an active microtubule depolymerase and mainly distributed on vesicles derived from the Golgi apparatus and destined for the cell surface. Thus, our results suggest that OsKinesin-13A utilizes its microtubule depolymerization activity to promote microtubule turnover, which may not only influence transverse orientation of cortical microtubules but also facilitate vesicle transport from the Golgi apparatus to the cell surface, and thus affects cellulose microfibril orientation and cell elongation.

Characterizing compression wood formed in radiata pine branches

The formation of reaction wood is an adaptive feature of trees in response to various mechanical forces. In gymnosperms, reaction wood consists of compression wood (CW) and opposite wood (OW) that are formed on the underside and upperside of bent trunks and branches. Although reaction wood formed in bent trunks has been extensively investigated, relatively little has been reported from conifer branches. In this study SilviScan® technology was used to characterize radiata pine branches at high resolution. Compared to OW formed in the branches, CW showed greater growth, darker colour, thicker tracheid walls, higher coarseness, larger microfibril angle (MFA), higher wood density, lower extensional stiffness and smaller internal specific surface area. However, tracheids of CW were similar to those of OW in their radial and tangential diameters. These results indicated that gravity influenced tracheid cell division and secondary wall formation but had limited impact on primary wall expansion. Furthermore, seasonal patterns of CW formation were not observed in the branches from cambial age 4 while earlywood and latewood were clearly separated in all rings of OW. The marked change of MFA during reaction wood formation suggested that branches could be ideal materials for further study of cellulose microfibril orientation.

Vibrational sum-frequency-generation (SFG) spectroscopy study of the structural assembly of cellulose microfibrils in reaction woods

The cellulose microfibril assemblies in secondary cell walls of tension wood and compression wood were studied with vibrational sum frequency generation (SFG) spectroscopy. The tension wood contains the gelatinous layer with highly-crystalline and highly-aligned cellulose microfibrils. The SFG spectral features of tension wood changed depending on the azimuth angle between the polarization of the incident IR beam and the preferential alignment axis of the cellulose microfibrils. The SFG spectra of the compression wood did not show any dependence on the azimuth angle, implying that the overall orientation of cellulose microfibrils in compression wood is not highly aligned. Instead, the decrease of cellulose content in compression wood brought about larger separation between cellulose microfibrils, which was manifested as changes in CH2/OH intensity ratio in SFG spectra. These results implied that SFG spectral features are sensitive to cellulose microfibril alignments and inter-fibrillar separations.

Seasonal and clonal variation in cellulose microfibril orientation during cell wall formation of tracheids in Cryptomeria japonica

To investigate the biological mechanism by which trees control the changes in microfibril (MF) orientation among secondary cell wall layers of conifer tracheids, we studied seasonal variation in the orientation of newly deposited MFs during tracheid cell wall development in Japanese cedar (Cryptomeria japonica D. Don) trees growing in Central Japan (36°36'N, 140°39'E). Sample blocks were repeatedly collected from four 16-year-old clones of different origins during the growing season of 2010 to investigate the hypotheses that changes in cellulose MF orientation between wall layers exhibited seasonal and clonal differences. The progressive change in the orientation of newly deposited MFs on the primary and secondary cell wall layers of tracheids was detected by field-emission-scanning electron microscopy. Tracheid production and differentiation was studied by light microscopy. We observed a decreasing trend in the orientation of deposited MFs from earlywood to latewood in the S2 and S1 layers, where MFs appeared in a Z-helix. In contrast, no seasonal pattern in the orientation of the MFs in the S-helix was observed. Minor clonal variation was observed in the phenology of tracheid production and differentiation. We concluded that a seasonal decreasing trend in the orientation of the MFs in the Z-helix in S1 and S2 was present, whereas the MFs in other layers exhibited minor random variations. Thus, the orientation of the MFs in S2 was affected by seasonal factors, whereas the MFs in other layers were more intrinsically controlled. The within-ring variations in the MF orientation and thus the resulting average MF angle might also be related to genotypic differences in the tracheid production and differentiation rate. However, our results do not exclude other intrinsic and environmental regulations in the change in MF orientation, which remains a topic for future studies.