Orientation Of Microfibrils Research Articles

Effect of microwave irradiation on the microfibril orientation in Phyllostachys iridescens bamboo

Taking small-diameter Phyllostachys iridescens bamboo as the research subject, the morphology of bamboo fiber cell walls was observed using scanning electron microscopy, atomic force microscopy, and polarized light microscopy, and a qualitative analysis was performed. X-ray diffraction, synchrotron radiation wide-angle X-ray scattering, and small-angle X-ray scattering were used to quantitatively study changes in the microfibril orientation angle and orientation degree of bamboo, revealing the changes in the bamboo fiber wall morphology and microfibril orientation before and after microwave irradiation treatment. The results showed that the cell wall surface of the samples became smoother and more even after microwave irradiation treatment. Before treatment, the microfibril angle of the samples was 15.87°, with an orientation degree of 0.94 and an average micropore length of 29.3 mm. After treatment, the microfibril angle decreased to 13.18°, the orientation degree increased to 0.97, and the average micropore length increased to 59.7 mm. The microfibril angle decreased by 2.68°, the orientation degree of samples increased by 0.03, and the average micropore length increased by 30.4 mm after microwave irradiation treatment. These results indicate that microwave irradiation treatment reduces the microfibril angle and increases the orientation degree in small-diameter bamboo. This provides a theoretical foundation for the rational and efficient utilization of Phyllostachys iridescens bamboo.

Ultra-microstructure analysis and model development of bamboo fiber cell walls

Bamboo fiber cell walls are crucial mechanical load-bearing units in plants, significantly influencing their physical and mechanical properties. This study explored the multi-layered structure and microfibril orientation of typical moso bamboo fiber with two broad layers. The microfibrils in the primary cell wall exhibited a disordered mesh-like structure with a microfibril angle (MFA) of 90°. The alternating narrow and broad layers in the secondary walls constituted the growth cycle layer together. Within this structure, the microfibrils were oriented in both Z-helices and S-helices. In the narrow layers, the MFA transitioned gradually from outer to inner layers, ranging from approximately 70° to 30°. Conversely, the broad layers had smaller MFA, ranging from about 5° to 20°. Finally, a typical model of multi-layered microfibril orientation of 4-year-old moso bamboo fiber cells was proposed.

NST3 induces ectopic transdifferentiation, forming secondary walls with diverse patterns and composition in Arabidopsis thaliana.

The master transcription factor NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3), also known as SND1, plays a pivotal role in regulating secondary cell wall (SCW) development in interfascicular and xylary fibers in Arabidopsis thaliana. Despite progress in understanding SCW assembly in xylem vessel-like cells, the mechanisms behind its assembly across different cell types remain unclear. Overexpressing NST3 or its homolog NST1 leads to reduced fertility, posing challenges for studying their impact on secondary wall formation. This study aimed at developing a tightly regulated dexamethasone (DEX)-inducible expression system for NST3 and NST1 to elucidate the structure and assembly of diverse SCWs. Using the DEX-inducible system, we characterized ectopically formed SCWs for their diverse patterns, mesoscale organization, cellulose microfibril orientation, and molecular composition using spinning disk confocal microscopy, field emission scanning electron microscopy (FESEM), vibrational sum-frequency generation (SFG) spectroscopy and, histochemical staining and time-of-flight secondary ion mass spectrometry (ToF-SIMS), respectively. Upon DEX treatment, NST3 and NST1 transgenic hypocotyls underwent time-dependent transdifferentiation, progressing from protoxylem-like to metaxylem-like cells. NST3-induced plants exhibited normal growth but had rough secondary wall surfaces with delaminating S2 and S3 layers. Mesoscale examination of induced SCWs in epidermal cells revealed that macrofibril thickness and orientation were comparable to xylem vessels, while wall thickness resembled that of interfascicular fibers. Additionally, induced epidermal cells formed SCWs with altered cellulose and lignin contents. These findings suggest NST3 and/or NST1 induce SCWs with shared characteristics of both xylem and fiber-like cells forming loosely arranged cell wall layers and cellulose organized at multiple angles relative to the cell growth axis and with varied cellulose and lignin abundance. This inducible system opens avenues to explore ectopic SCWs for bioenergy and bioproducts, offering valuable insights into SCW patterning across diverse cell types and developmental stages.

Effect of hygro-mechanical treatment combined with saturated steam on bamboo cell wall: Structural, chemical, and hygroscopic properties

Bamboo has shown great potential in various fields such as construction, furniture, and decoration due to its fast growth rate, short degradation cycle, strong carbon sequestration ability, and excellent mechanical properties. The effects of hygro-mechanical treatment combined with saturated steam (SC treatment) on bamboo cell walls are less studied. This study explored the effects of SC treatment at different temperatures (140°C, 160°C, 180°C) on the cell wall structure, chemical composition, and hygroscopic properties of bamboo. The study utilized Fourier transform infrared spectroscopy (FTIR) and Confocal Raman microscopy (CRM) to analyze the chemical properties of bamboo, X-ray diffraction (XRD) to analyze the crystal structure, Small Angle X-ray Scattering (SAXS) to analyze the microstructure, Brunauer-Emmett-Teller (BET) to analyze porosity, and Dynamic Vapor Sorption (DVS) to assess humidity adsorption. The lignin and hemicellulose in the bamboo were degraded under saturated steam treatment, leading to reduced stability of the bamboo fiber structure. Compression under these conditions severely damages the orderly arrangement of the fiber structure, resulting in a decrease in the microfibril orientation. The reduction in orientation significantly affected the moisture transfer rate in bamboo and the micromechanical properties of the fiber cells. Simultaneously, the reduction of lignin and hemicellulose decreases the hygroscopic properties of bamboo. The results of this study indicate that SC treatment significantly improves the crystallinity and dimensional stability of bamboo while reducing its porosity, hygroscopicity, and micromechanical properties. Among the three experimental groups, the 160°C saturation steam-compression treatment was considered the best method for significantly improving bamboo performance while maintaining structural integrity.

Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Correlating macroscopic plant growth parameters to nanomechanical properties of cellulose microfibrils

The plant cell wall, a vital component in providing structural integrity and facilitating growth, comprises cellulose microfibrils among its major constituents. This study employed Atomic Force Microscopy (AFM) to investigate the intricate relationship between genetic mutation, cellulose microfibril organization, nanomechanical properties of cellulose microfibrils and plant growth. Focusing on the Arabidopsis thaliana wild type (WT) and ixr1–2 mutant population (known for resistance to herbicide ISOXABEN), we utilized AFM to scrutinize cellulose microfibrils on the newly synthesized cell wall in 5-day-old dark-grown hypocotyls. Our macroscopic analysis revealed significant differences in plant growth, prompting a detailed examination at the nanoscale using AFM to discover if the macroscopic disparity between these two populations gets translated in structural details, orientation, and mechanical properties of cellulose microfibrils at the nanoscale too. AFM analysis highlighted distinct organizational disparities in cellulose microfibrils between the WT and mutant population. Our results revealed that the WT manifests a more aligned and oriented microfibril structure in contrast to the mutant population that shows significantly less aligned cellulose microfibrils in the plant growth direction. Also, the WT and mutant population demonstrate nuanced differences in height, width, roughness, deformation, and stiffness. The observed nanoscale alterations in microfibril structure and nano-mechanical properties contribute to an improved understanding of the intricate dynamics governing plant cell wall structure and its pivotal role in growth and development.

Characterization of the fiber-like cortical cells in moss gametophytes.

Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-β-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-β-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Cortical microtubule dynamics during reaction wood formation ensures context-appropriate cellulose microfibril angle in woody trees

Key messageCortical microtubule arrays are the primary mechanism for guiding the re-orientation of cellulose microfibrils and determining MFA in secondary cell wall of wood fibre and tracheid cells in reaction wood.Microtubules are directly and indirectly involved in guiding cellulose synthase complexes (CSCs) through the plasma membrane. The angle of cellulose deposition is a critical response to environmental signals and/or stress conditions, and particularly crucial during reaction wood formation, a process in which woody plants deposit additional cell wall material to counteract gravitational forces. Tubulin genes are upregulated in response to gravitational stimulus during reaction wood formation, which can result in changes to microtubule assembly. In this study, microtubules were visualised in three woody tree species (two angiosperms: Eucalyptus globulus Labill., Populus alba L., and one gymnosperm: Pinus radiata D. Don.) using immunofluorescence to quantitatively evaluate microtubule organisation during reaction wood formation. Our results suggest that reorientation of the cortical microtubule array affects secondary cell wall deposition, even across different types of reaction wood, by ensuring context-appropriate orientation of cellulose microfibrils and determining MFA in wood cells. Pharmacological studies conducted on in vitro cultured stem segments or in vivo during reaction wood formation corroborated these important roles for microtubules during wood development. This study starts to unveil the role of tubulins during wood formation by exploring cortical microtubule array organisation in trees subjected to gravitational stimulus and it sheds light on cellular and molecular mechanisms behind cellulose deposition in tree species.

Growth and tension in explosive fruit

Exploding seed pods of the common weed Cardamine hirsuta have the remarkable ability to launch seeds far from the plant. The energy for this explosion comes from tension that builds up in the fruit valves. Above a critical threshold, the fruit fractures along its dehiscence zone and the two valves coil explosively, ejecting the seeds. A common mechanism to generate tension is drying, causing tissues to shrink. However, this does not happen in C.hirsuta fruit. Instead, tension is produced by active contraction of growing exocarp cells in the outer layer of the fruit valves. Exactly how growth causes the exocarp tissue to contract and generate pulling force is unknown. Here we show that the reorientation of microtubules in the exocarp cell cortex changes the orientation of cellulose microfibrils in the cell wall and the consequent cellular growth pattern. We used mechanical modeling to show how tension emerges through growth due to the highly anisotropic orientation of load-bearing cellulose microfibrils and their effect on cell shape. By explicitly defining the cell wall as multi-layered in our model, we discovered that a cross-lamellate pattern of cellulose microfibrils further enhances the developing tension in growing cells. Therefore, the interplay of cell wall properties with turgor-driven growth enables the fruit exocarp to generate sufficient tension to power explosive seed dispersal.

A 3D multi-scale hygro-mechanical model of oak wood

A multi-scale framework is proposed for the prediction of the macroscopic hygro-elastic properties of oak wood. The distinctive features of the current multi-scale approach are that: (i) Four different scales of observation are considered, which enables the inclusion of heterogeneous effects from the nano-, micro-, and meso-scales in the effective constitutive behavior of oak at the macro-scale, (ii) the model relies on three-dimensional material descriptions at each considered length scale, and (iii) a moisture-dependent constitutive assumption is adopted at the nano-scale, which allows for recovering the moisture dependency of the material response at higher scales of observation. In the modeling approach, oak wood is assumed as homogeneous at the macro-scale. The meso-scale description considers the cellular structure of individual growth rings with three different densities. At the micro-scale, the heterogeneous nature of cell walls is described by the characteristics of the primary and secondary cell wall layers. Finally, the nano-scale response is determined by cellulose micro-fibrils embedded in a matrix of hemicellulose and lignin. The oak properties at the four length scales are connected via a three-level homogenization procedure, for which, depending on the geometry of the fine-scale configuration, an asymptotic homogenization procedure or Voigt averaging procedure is applied at each level to determine the effective hygro-elastic properties at the corresponding coarse scale. In addition, the moisture adsorption isotherms at each scale are constructed from a volume-weighted averaging of the moisture adsorption characteristics at the scale below. The computational results demonstrate that the macro-scale moisture-dependent, hygro-elastic behavior of oak wood is predicted realistically, thereby revealing the influence of the material density, the micro-fibril orientation, and the hygro-elastic properties from the underlying scales. The computed macro-scale properties of oak are in good agreement with experimental data reported in the literature.

Recent genome resequencing paraded COBRA-Like gene family roles in abiotic stress and wood formation in Poplar.

A cell wall determines the mechanical properties of a cell, serves as a barrier against plant stresses, and allows cell division and growth processes. The COBRA-Like (COBL) gene family encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein that controls cellulose deposition and cell progression in plants by contributing to the microfibril orientation of a cell wall. Despite being studied in different plant species, there is a dearth of the comprehensive global analysis of COBL genes in poplar. Poplar is employed as a model woody plant to study abiotic stresses and biomass production in tree research. Improved genome resequencing has enabled the comprehensive exploration of the evolution and functional capacities of PtrCOBLs (Poplar COBRA-Like genes) in poplar. Phylogeny analysis has discerned and classified PtrCOBLs into two groups resembling the Arabidopsis COBL family, and group I genes possess longer proteins but have fewer exons than group II. Analysis of gene structure and motifs revealed PtrCOBLs maintained a rather stable motif and exon-intron pattern across members of the same group. Synteny and collinearity analyses exhibited that the evolution of the COBL gene family was heavily influenced by gene duplication events. PtrCOBL genes have undergone both segmental duplication and tandem duplication, followed by purifying selection. Promotor analysis flaunted various phytohormone-, growth- and stress-related cis-elements (e.g., MYB, ABA, MeJA, SA, AuxR, and ATBP1). Likewise, 29 Ptr-miRNAs of 20 families were found targeting 11 PtrCOBL genes. PtrCOBLs were found localized at the plasma membrane and extracellular matrix, while gene ontology analysis showed their involvement in plant development, plant growth, stress response, cellulose biosynthesis, and cell wall biogenesis. RNA-seq datasets depicted the bulk of PtrCOBL genes expression being found in plant stem tissues and leaves, rendering mechanical strength and rejoinders to environmental cues. PtrCOBL2, 3, 10, and 11 manifested the highest expression in vasculature and abiotic stress, and resemblant expression trends were upheld by qRT-PCR. Co-expression network analysis identified PtrCOBL2 and PtrCOBL3 as hub genes across all abiotic stresses and wood developing tissues. The current study reports regulating roles of PtrCOBLs in xylem differentiating tissues, tension wood formation, and abiotic stress latency that lay the groundwork for future functional studies of the PtrCOBL genes in poplar breeding.

Structure of cellulose in birch phloem fibres in tension wood: an X-ray nanodiffraction study

BackgroundTo gain a better understanding of bark layer structure and function, especially of the phloem fibres and their contribution to the posture control of trees, it is important to map the structural properties of these cells. The role of bark can also be linked to the reaction wood formation and properties which are essential when it comes to studying the questions related to tree growth. To offer new insights into the role of bark in the postural control of trees, we studied the micro- and nanoscale structures of the phloem and its nearest layers. This study is the first time, in which phloem fibres in trees have been extensively examined using X-ray diffraction (XRD).We determined the orientation of cellulose microfibrils in phloem fibres of Silver birch saplings by using scanning synchrotron nanodiffraction. The samples consisted of phloem fibres extracted from tension, opposite and normal wood (TW, OW, NW).ResultsUsing scanning XRD, we were able to obtain new information about the mean microfibril angle (MFA) in cellulose microfibrils in phloem fibres connected to reaction wood. A slight but consistent difference was detected in the average MFA values of phloem fibres between the TW and OW sides of the stem. Using scanning XRD, different contrast agents (intensity of the main cellulose reflection or calcium oxalate reflection, mean MFA value) were used to produce 2D images with 200 nm spatial resolution.ConclusionsBased on our results, the tension wood formation in the stem might be related to the structure and properties of phloem fibres. Thus, our results suggest that the nanostructure of phloem fibres is involved in the postural control of trees containing tension and opposite wood.

Combined X-ray diffraction tomography imaging of tension and opposite wood tissues in young hybrid aspen saplings

Combining spatially localized X-ray diffraction (XRD) with X-ray microtomography (XMT) enables the mapping of the micro- and nanoscale structures simultaneously. The combination of these methods results in a powerful tool when considering the structural studies of hierarchical materials, allowing one to couple the relationships and connections of the structures at various scales. In this study, XMT was used to map the anatomy and cellular structures in 3D in tension and opposite wood with 1.5 µm resolution, while XRD was used to determine the cellulose crystallite widths and microfibril orientations with 100 µm spatial resolution within the same tissues. Tension wood (TW) has an important biological function with clearly distinct properties to opposite (OW) and normal wood, e.g. differing cellular structures with a higher cellulose content. This is the first study of very young hybrid aspen saplings (1-month-old) using the combined diffraction tomography method. The TW tissues could be identified from the OW tissues based on both the XMT and XRD results: TW had a higher average size of the cellulose crystallites and smaller mean microfibril angles (mMFA) than those in OW. With the XRD data, we were able to reconstruct the images of the cross sections of the saplings using the structural parameters (cellulose crystallite width and mMFA) as contrast mechanisms. As far as the authors know, there are no previous studies with images on any TW samples using the XRD-based contrast. Home laboratory bench-top set-up offers its advantages for these studies, considering the number of samples characterized, time-dependent studies and larger field of views.

Effects of microwave drying on the cell wall structures of round bamboo

Effects of microwave irradiation on bamboo cell wall structures were studied. Microwave treatment resulted in samples exhibiting smoother surfaces, deformed parenchyma cells, and increased porosity. Microwave treatment altered microfibril orientation through the formation of hydrogen bonds. The peak at 3440 cm-1, assigned to O-H stretching, decreased. Meanwhile, intermolecular and intramolecular bonds were formed, increasing the uniformity of microfibrils. Condensation reactions in surface hydroxyl groups and the formation of intramolecular hydroxyl groups in the amorphous region under microwave irradiation also improved the fiber arrangement uniformity. After treatment, the HCH and HOC bonds were reduced and the ester bonds were broken down. The methoxy and aromatic ring hydroxyl groups were oxidized, as indicated by the increase in the absorption peak at 148 ppm. The hydroxyl groups in the amorphous region near the fiber surface decreased, as did hemicellulose content, but there was an increase in secondary crystalline fibers.

Anisotropic cell growth and cell wall structure in lenticular cell of Valonia utricularis (Ulvophyceae)

SummaryDuring cell division of the giant‐celled green alga, Valonia utricularis, a lenticular cell is newly formed, which grows from disc‐shaped to globular to obovoid. During the early developmental stages of growth, the cell surface shows a remarkable outward protrusion. In the present study, the anisotropy of cell growth, i.e. the difference between cell surface extension in meridional and radial orientation, was investigated by analyzing the movement of the surface markers in a living cell. Growth was isotropic around the cell zenith but of two different kinds of anisotropic growth in other regions; radial extension was dominant in cell periphery and meridional extension in intermediate regions between zenith and periphery. Moreover, local orientation of cellulose microfibrils was observed on the inner surface of the cell wall during different stages of early development in lenticular cell using an atomic force microscope. Cellulose microfibrils showed meridional orientation overall and this phenomenon was most remarkable in the periphery of the cell, suggesting the possibility of cellulose microfibrils promoting radial extension of cells by suppressing meridional extension of cell wall.

Measurement of microfibril angle in plant fibres: Comparison between X-ray diffraction, second harmonic generation and transmission ellipsometry microscopies

The orientation of cellulose microfibrils within plant fibres is one of the main factors influencing their mechanical properties. As plant fibres are more and more used as reinforcement for agro-composites, their mechanical properties have a strong influence on the final composite properties. It is, therefore, of interest to obtain reliable information about the microfibril angle (MFA) to better support the choice of fibres depending on the product requirements. In the present study, the reliability and specificities of three non-destructive methods that allow analysis on the same fibre glued on a holder; X-ray diffraction (XRD), second harmonic generation (SHG) and transmission ellipsometry (TE) microscopy; are investigated. Three types of plant fibres, with both low (nettle), and high (cotton, sisal) MFA values, are compared and their geometry and biochemical composition are characterised. The results obtained on the same fibre confirm that MFA analysis remains tedious and that despite their limitations, the methods are complementary depending on the information requested. Indeed, SHG is recommended for direct, qualitative and plane-selective mapping of heterogeneities in macrofibril orientations at various depths. However, reliable quantitative results with SHG depend on the initial image quality and could benefit from further image processing refinement. On the contrary, XRD and TE measure MFAs over the entire fibre thickness and provide variations along the fibres if a sufficient optical/spatial resolution is reached. Regarding the characterization of intrinsic defects in plant fibres, both SHG and TE suffer from uncertainties induced by the disorganization of the microfibril network and the lack of symmetry between the front and back fibre walls. Finally, all techniques prove to be dependant on the initial fibre alignment and geometry (i.e. twisting, double fibre configuration or form factor) which vary along the fibre length and should be carefully taken into account.

Water sorption in wood cell walls–data exploration of the influential physicochemical characteristics

The material properties of wood are intimately tied to the amount of moisture contained in the wood cell walls. The moisture content depends on the environmental conditions, i.e. temperature and relative humidity, but also on material characteristics of the wood itself. The exact mechanisms governing moisture equilibrium between wood cell walls and environmental conditions remain obscure, likely because multiple material characteristics have been proposed to be involved. In this study, we used a data exploration approach to illuminate the important wood characteristics determining the cell wall moisture content in the full moisture range. Specimens of nine different wood species (two softwoods and seven hardwoods) were examined in terms of their material characteristics at multiple scales and their cell wall moisture content was measured in equilibrium with both hygroscopic conditions and at water-saturation. By statistical analysis, the chemical composition was found to be the most important predictor of the cell wall moisture content in the full moisture range. For the other wood characteristics the importance differed between the low moisture range and the humid and saturated conditions. In the low moisture range, the cellulose crystallinity and hydroxyl accessibility were found to be important predictors, while at high moisture contents the microfibril orientation in the S1 and S3 layers of the cell walls was important. Overall, the results highlighted that no single wood characteristic were decisive for the cell wall moisture content, and each of the predictors identified by the analysis had only a small effect in themselves on the cell wall moisture content. Wood characteristics with a major effect on the cell wall moisture content were, therefore, not identified..

Detecting the orientation of newly-deposited crystalline cellulose with fluorescent CBM3

Cellulose microfibril patterning influences many of the mechanical attributes of plant cell walls. We developed a simple, fluorescence microscopy-based method to detect the orientation of newly-synthesized cellulose microfibrils in epidermal peels of onion and Arabidopsis. It is based on Alexa Fluor 488-tagged carbohydrate binding module 3a (CBM3a) from Clostridium thermocellum which displayed a nearly 4-fold greater binding to cell walls at pH 5.5 compared with pH 8. Binding to isolated cellulose did not display this pH dependence. At pH 7.5 fibrillar patterns at the surface of the epidermal peels were visible, corresponding to the directionality of surface cellulose microfibrils, as verified by atomic force microscopy. The fibrillar pattern was not visible as the labeling intensity increased at lower pH. The pH of greatest cell wall labeling corresponds to the isoelectric point of CBM3a, suggesting that electrostatic forces limit CBM3a penetration into the wall. Consistent with this, digestion of the wall with pectate lyase to remove homogalacturonan increased labeling intensity. We conclude that electrostatic interactions strongly influence labeling of cell walls with CBM3 and potentially other proteins, holding implications for any work that relies on penetration of protein probes such as CBMs, antibodies, or enzymes into charged polymeric substrates.

Polarized Raman spectroscopy for determining the orientation of cellulose microfibrils in wood cell wall

Polarized Raman spectroscopy for determining the orientation of cellulose microfibrils in wood cell wall

Chemonastic Stalked Glands in the Carnivorous Rainbow Plant Byblis gigantea LINDL. (Byblidaceae, Lamiales).

Carnivorous rainbow plants (Byblis, Byblidaceae, Lamiales) possess sticky flypaper traps for the capture, retention, and digestion of prey (mainly small insects). The trapping system is based on a multitude of millimeter-sized glandular trichomes (also termed stalked glands), which produce adhesive glue drops. For over a century, the trapping system of Byblis was considered passive, meaning that no plant movement is involved. Recently, a remarkable discovery was made: the stalked glands of Byblis are indeed capable of reacting to chemical (protein) stimuli with slow movement responses. This prompted us to investigate this phenomenon further with a series of experiments on the stimulation, kinematics, actuation, and functional morphology of the stalked glands of cultivated Byblis gigantea plants. Measured stalked gland lengths and densities on the trap leaves are similar to the data from the literature. Motion reactions could only be triggered with chemical stimuli, corroborating the prior study on the stalked gland sensitivity. Reaction time (i.e., time from stimulation until the onset of motion) and movement duration are temperature-dependent, which hints towards a tight physiological control of the involved processes. The stalked gland movement, which consist of a sequence of twisting and kinking motions, is rendered possible by the components of the stalk cell wall and is furthermore anatomically and mechanically predetermined by the orientation of cellulose microfibrils in the cell wall. Successive water displacement processes from the stalk cell into the basal cells actuate the movement. The same kinematics could be observed in stalked glands drying in air or submersed in a saturated salt solution. Stimulated and dried stalked glands as well as those from the hypertonic medium were capable of regaining their initial shape by rehydration in water. However, no glue production could be observed afterwards. The long-time overlooked chemonastic movements of stalked glands may help Byblis to retain and digest its prey; however, further research is needed to shed light on the ecological characteristics of the rainbow plant’s trapping system.