Chemical Composition Of Cell Walls Research Articles

Variation pattern in the macromolecular (cellulose, hemicelluloses, lignin) composition of cell walls in Pinus tabulaeformis tree trunks at different ages as revealed using multiple techniques

The plant cell wall is a complex, heterogeneous structure primarily composed of cellulose, hemicelluloses, and lignin. Exploring the variations in these three macromolecules over time is crucial for understanding wood formation to enhance chemical processing and utilization. Here, we comprehensively analyzed the chemical composition of cell walls in the trunks of Pinus tabulaeformis using multiple techniques. In situ analysis showed that macromolecules accumulated gradually in the cell wall as the plant aged, and the distribution pattern of lignin was opposite that of polysaccharides, and both showed heterogenous distribution patterns. In addition, gel permeation chromatography (GPC) results revealed that the molecular weights of hemicelluloses decreased while that of lignin increased with age. Two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) analysis indicated that hemicelluloses mainly comprised galactoglucomannan and arabinoglucuronoxylan, and the lignin types were mainly comprised guaiacyl (G) and p-hydroxyphenyl (H) units with three main linkage types: β-O-4, β-β, and β-5. Furthermore, the C-O bond (β-O-4) signals of lignin decreased while the C-C bonds (β-β and β-5) signals increased over time. Taken together, these findings shed light on wood formation in P. tabulaeformis and lay the foundation for enhancing the processing and use of wood and timber products.

Innovative chitin-glucan based material obtained from mycelium of wood decay fungal strains

Fungi are an alternative source to animal-based chitin. In fungi, chitin fibrils are strongly interconnected and bound with glucans that justify the unique matrix. The present study aimed to extract chitin and glucans from the mycelium of several wood decay fungal strains in order to obtain flexible materials and to check correlations between chitin content and the mechanical properties of these materials. Five strains were chosen in consideration of their different cell wall chemical composition (high content of α-glucans, β-glucans or chitin) to evaluate how these differences could influence the mechanical and chemical characteristics of the material. The fungal strains were cultivated in liquid-submerged dynamic fermentation (both flasks and bioreactor). Chitin and glucans were crosslinked with acetic acid and plasticized with glycerol to obtain flexible sheets. Abortiporus biennis, Fomitopsis iberica and Stereum hirsutum strains were found to adapt to produce material with adequate flexibility. The obtained materials were characterized by Thermogravimetric analysis (TGA) for the understanding of the material composition. The material obtained from each species was mechanically tested in terms of tear strength, elongation at break, and Young's modulus.

Simple and Rapid Discrimination of Methicillin-Resistant Staphylococcus aureus Based on Gram Staining and Machine Vision.

Methicillin-resistant Staphylococcus aureus (MRSA) is a clinical threat with high morbidity and mortality. Here, we describe a new simple, rapid identification method for MRSA using oxacillin sodium salt, a cell wall synthesis inhibitor, combined with Gram staining and machine vision (MV) analysis. Gram staining classifies bacteria as positive (purple) or negative (pink) according to the cell wall structure and chemical composition. In the presence of oxacillin, the integrity of the cell wall for methicillin-susceptible S. aureus (MSSA) was destroyed immediately and appeared Gram negative. In contrast, MRSA was relatively stable and appeared Gram positive. This color change can be detected by MV. The feasibility of this method was demonstrated in 150 images of the staining results for 50 clinical S. aureus strains. Based on effective feature extraction and machine learning, the accuracies of the linear linear discriminant analysis (LDA) model and nonlinear artificial neural network (ANN) model for MRSA identification were 96.7% and 97.3%, respectively. Combined with MV analysis, this simple strategy improved the detection efficiency and significantly shortened the time needed to detect antibiotic resistance. The whole process can be completed within 1 h. Unlike the traditional antibiotic susceptibility test, overnight incubation is avoided. This new strategy could be used for other bacteria and represents a new rapid method for detection of clinical antibiotic resistance. IMPORTANCE Oxacillin sodium salt destroys the integrity of the cell wall of MSSA immediately, appearing Gram negative, whereas MRSA is relatively stable and still appears Gram positive. This color change can be detected by microscopic examination and MV analysis. This new strategy has significantly reduced the time to detect resistance. The results show that using oxacillin sodium salt combined with Gram staining and MV analysis is a new, simple and rapid method for identification of MRSA.

Protoplast Preparation for Algal Single-Cell Omics Sequencing.

Single-cell sequencing (SCS) is an evolutionary technique for conducting life science research, providing the highest genome-sale throughput and single-cell resolution and unprecedented capabilities in addressing mechanistic and operational questions. Unfortunately, the current SCS pipeline cannot be directly applied to algal research as algal cells have cell walls, which makes RNA extraction hard for the current SCS platforms. Fortunately, effective methods are available for producing algal protoplasts (cells without cell walls), which can be directly fed into current SCS pipelines. In this review, we first summarize the cell wall structure and chemical composition of algal cell walls, particularly in Chlorophyta, then summarize the advances made in preparing algal protoplasts using physical, chemical, and biological methods, followed by specific cases of algal protoplast production in some commonly used eukaryotic algae. This review provides a timely primer to those interested in applying SCS in eukaryotic algal research.

Modification of Poplar Wood via Polyethylene Glycol Impregnation Coupled with Compression

Wood permeability and compressibility are affected by cell wall structure and chemical composition. These properties can be improved by appropriate wood pretreatments. Low-density poplar wood was converted to a more dense structure by the following steps: First, lignin and hemicellulose were removed using a mixture of NaOH and Na2SO3. Second they were impregnated with polyethylene glycol (PEG, mean molecular weight of 1200), nano-SiO2, and a silane coupling agent at atmospheric temperature and pressure. Finally, impregnated wood was compressed at 150 °C. Results showed that the tracheid lumens on the transverse section of the compressed wood almost vanished. Specifically, the lumens in the wood cells, especially those that were compressed, were almost completely filled with PEG. In FTIR, the asymmetric absorption peaks of Si–O–Si at 1078–1076 cm−1 were clearly observed, which confirms the existence of bonding between nano-SiO2 and wood. The highest melting enthalpy and crystallization enthalpy showed a heat storage capacity of modified wood, which were 20.7 and 9.8 J/g, respectively. Such phase change capabilities may have potential applications in regulating the rate of change of room temperature. In summary, the modified wood could be utilized as material for construction to conserve energy.

Expression of Exogenous GFP-CesA6 in Tobacco Enhances Cell Wall Biosynthesis and Biomass Production.

Simple SummaryCellulose is synthesized at the plasma membrane by an enzymatic complex constituted by different cellulose synthase (CesA) proteins. The overexpression of CesA genes has been assessed for increasing cellulose biosynthesis and plant biomass. In this study, we analyzed transgenic tobacco plants (F31 line), stably expressing the Arabidopsis CesA6 fused to GFP, for possible variations in the cellulose biosynthesis. We found that F31 plants were bigger than the wild-type (wt), showing significant increases of stem height, root length, and leaf area. They bloomed about 3 weeks earlier and yielded more flowers and seeds than wt. In the F31 leaves, the expression of the exogenous GFP-CesA6 prompted the overexpression of all CesAs involved in the synthesis of primary cell wall cellulose and of other proteins responsible for plant cell wall building and remodeling. Instead, secondary cell wall CesAs were not affected. In the F31 stem, showing a 3.3-fold increase of the secondary xylem thickness, both primary and secondary CesAs expression was differentially modulated. Significantly, the amounts of cellulose and matrix polysaccharides increased in the transformed seedlings. The results evidence the potentiality to overexpress primary CesAs in tobacco for biomass production increase.Improved cellulose biosynthesis and plant biomass represent important economic targets for several biotechnological applications including bioenergy and biofuel production. The attempts to increase the biosynthesis of cellulose by overexpressing CesAs proteins, components of the cellulose synthase complex, has not always produced consistent results. Analyses of morphological and molecular data and of the chemical composition of cell walls showed that tobacco plants (F31 line), stably expressing the Arabidopsis CesA6 fused to GFP, exhibits a “giant” phenotype with no apparent other morphological aberrations. In the F31 line, all evaluated growth parameters, such as stem and root length, leaf size, and lignified secondary xylem, were significantly higher than in wt. Furthermore, F31 line exhibited increased flower and seed number, and an advance of about 20 days in the anthesis. In the leaves of F31 seedlings, the expression of primary CesAs (NtCesA1, NtCesA3, and NtCesA6) was enhanced, as well as of proteins involved in the biosynthesis of non-cellulosic polysaccharides (xyloglucans and galacturonans, NtXyl4, NtGal10), cell wall remodeling (NtExp11 and XTHs), and cell expansion (NtPIP1.1 and NtPIP2.7). While in leaves the expression level of all secondary cell wall CesAs (NtCesA4, NtCesA7, and NtCesA8) did not change significantly, both primary and secondary CesAs were differentially expressed in the stem. The amount of cellulose and matrix polysaccharides significantly increased in the F31 seedlings with no differences in pectin and hemicellulose glycosyl composition. Our results highlight the potentiality to overexpress primary CesAs in tobacco plants to enhance cellulose synthesis and biomass production.

24-Epibrassinolide and 2,6-Dichlorobenzonitrile Promoted Celery Petioles and Hypocotyl Elongation by Altering Cellulose Accumulation and Cell Length

BRs (brassinosteroids), an endogenous hormone in plants, regulate cellulose accumulation, cell elongation and plant growth. Propiconazole (PCZ) is an effective inhibitor of BR biosynthesis. DCB (2,6-Dichlorobenzonitrile) can inhibit the synthesis of cellulose and affects the chemical composition of cell walls. Celery is one important leafy vegetable of the Apiaceae family with rich dietary fiber (including cellulose). The petioles length, leaf blades number and cellulose content determine the yield and quality of celery. The family members of AgCESAs are related to cellulose biosynthesis in higher plants. To investigate the effects of BRs, PCZ and DCB on the growth of celery, celery cv. ‘Jinnan Shiqin’ plants were treated with 24-epibrassinolide (24-EBL, most active form of BRs), PCZ and DCB, respectively. The results showed that exogenous application of BRs up-regulated the expression of AgCESAs genes and accumulated more cellulose in celery. The length of petioles and number of leaf blades in celery plants applied with exogenous BRs (1.24 × 10−6 mol/L 24-EBL) were increased 2.16 and 1.37 times of that in the control. The addition of PCZ inhibited the effects of exogenous BRs application. The lengths of hypocotyl and hypocotyl cells of celery plants treated with BRs were longer than that of the control. Under DCB treatments, the expression levels of AgCESAs genes in celery petioles and leaf blades were down-regulated compared with the control, and the celery plants showed decreased cellulose content, shorter petiole length and fewer leaf blades. The length of hypocotyl and hypocotyl cells of celery treated with DCB were shorter than that of the control. This study provided a reference for the functions of BRs and DCB on the growth and development of celery.

The removal of iron from waterlogged archaeological wood: efficacy and effects on the room temperature wood properties

ABSTRACT Present results provided new information on the practice of removing iron from waterlogged wood using chelating agents. Two series of samples were selected: small waterlogged wood samples of Quercus spp., Pinus sylvestris L. and Pinus pinaster Aiton, and a portion from the dismantled shipwreck Huguangjiao I. Iron extractions were carried out using disodium ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). Samples were characterized by environmental scanning electron microscopy coupled with dispersive energy spectrometry, micro-Raman spectroscopy, atomic absorption spectroscopy, Fourier transform infrared spectroscopy and dynamic mechanical analysis (DMA), the latter used to evaluate viscoelastic properties. The extraction efficiency of both products was good, although it depended on the wood state of preservation, being higher in more decayed material. Still, the extraction procedures induced a limited effect on the decayed wood tissue, affecting both cell wall chemical composition and physical characteristics. Moreover, a significant difference existed between the two agents. In decayed hardwoods, they had an impact on both E’ (decrease) and tanδ (increase), mostly apparent for DTPA. The difference between DTPA and EDTA was also more evident when the frequency was varied in DMA tests. In contrast, the well-preserved softwood was not affected by the considered treatments.

BIOACTIVE COMPOUNDS AND ANTIOXIDANT ACTIVITY OF PERIPHERAL LAYERS OF SOFT WHEAT GROWN IN ALGERIA DURING SEED FILLING

Wheat bran is a by-product rich in bioactive compounds. However, little is known about the arrangement, composition and secondary metabolisms of the grain outer layers during grain development. To this end, the objective of this study is to evaluate the variations in cell wall structure and chemical composition of the outer layers during the cellularization phase, the actual filling of the grain and the maturation phase. Morphological and structural aspects were evaluated by scanning electron microscopy; phytochemical study also was carried out: Extracts from the peripheral layers (CP) of wheat grain (cultivar Triticum aestivum HD1220) were analyzed to determine: Total phenols content by Folin-Ciocâlteu method, flavonoids by aluminum trichloride and sodium hydroxide method; Antioxidant power was evaluated by free radical scavenging (DPPH) and iron reduction (FRAP). The results of histological analysis showed that the outer layers become thinner during grain development, the main tissues develop and differentiate synchronously; the phenolic content ranged from 47.58± 3.23 mg GAE/g to 108.67± 10.7 mg GAE/g wheat outer layers, the wheat outer layers contained 6.96±2.61-35.297±8.68 mg EAQ/g flavonoids. The contents of total phenols (TPC) and flavonoids (TFC) increased during development; the antioxidant activity, depended on the developmental phase of the peripheral layers; the antioxidant activity studied for 70% ethanolic extracts was highest in the peripheral layers of mature grains. These results underline that the peripheral layers of wheat bran derived from maternal tissues are constituted by a superposition of different tissues and are considered as an important source of bioactive compounds.

A Comparative Investigation on Structural and Chemical Differences between the Pith and Rind of Sunflower Stalk and Their Influences on Nanofibrillation Efficiency

The structure and chemical composition of cell walls play a vital role in the bioconversion and utilization of plants. In the present study, the cell wall structure and chemical composition of pith and rind from sunflower stalks were compared and correlated to their nanofibrillation efficiency with ultrasonic treatment. Mild chemical pretreatment using 1% or 4% NaOH without any bleaching process were applied prior to ultrasonication nanofibrillation. Significant structural and chemical differences were demonstrated between the pith and rind, with the former exhibiting a much lower lignin and hemicellulose contents, higher pectin, much looser cell structure and higher cell wall porosity than the latter. Alkaline treatment alone was sufficient to eliminate most of the hemicellulose and pectin from stalk pith, whereas only partial removal of hemicellulose and lignin was achieved for the woody rind part. After 30 min of ultrasonic treatment, the stalk pith exhibited fully defibrillated fibrils with a continuous and entangled micro/nanofibrillated network, whereas numerous micron-sized fiber and fragments remained for the rind. The results indicated that stalk pith is less recalcitrant and easier to be fibrillated with ultrasonication than rind, which must be correlated to their distinct differences in both structure and chemical composition.

Near-Infrared Spectroscopy can Predict Anatomical Abundance in Corn Stover

Feedstock heterogeneity is a key challenge impacting the deconstruction and conversion of herbaceous lignocellulosic biomass to biobased fuels, chemicals, and materials. Upstream processing to homogenize biomass feedstock streams into their anatomical components via air classification allows for a more tailored approach to subsequent mechanical and chemical processing. Here, we show that differing corn stover anatomical tissues respond differently to pretreatment and enzymatic hydrolysis and therefore, a one-size-fits-all approach to chemical processing biomass is inappropriate. To inform on-line downstream processing, a robust and high-throughput analytical technique is needed to quantitatively characterize the separated biomass. Predictive correlation of near-infrared spectra to biomass chemical composition is such a technique. Here, we demonstrate the capability of models developed using an “off-the-shelf,” industrially relevant spectrometer with limited spectral range to make strong predictions of both cell wall chemical composition and the relative abundance of anatomical components of the corn stover, the latter for the first time ever. Gaussian process regression (GPR) yields stronger correlations (average R2v = 88% for chemical composition and 95% for anatomical relative abundance) than the more commonly used partial least squares (PLS) regression (average R2v = 84% for chemical composition and 92% for anatomical relative abundance). In nearly all cases, both GPR and PLS outperform models generated using neural networks. These results highlight the potential for coupling NIRS with predictive models based on GPR due to the potential to yield more robust correlations.

Seasonal changes in cambium activity from active to dormant stage affect the formation of secondary xylem in Pinus tabulaeformis Carr.

Understanding the changing patterns of vascular cambium during seasonal cycles is crucial to reveal the mechanisms that control cambium activity and wood formation, but this area has been underexplored, especially in conifers. Here, we quantified the changing cellular morphology patterns of cambial zones during the active, transition and dormant stages. With the help of toluidine blue and periodic acid-Schiff staining to visualize cell walls and identify their constituents, we observed decreasing cambial cell layers, thickening of newly formed xylem cell walls and increased polysaccharide granules in phloem from June to the following March over the course of our collecting period. Pectin immunofluorescence showed that dormant-stage cambium can produce highly abundant de-esterified homogalacturonan and (1-4)-β-d-galactan epitopes, whereas active cambium can strong accumulate high methylesterified homogalacturonan. Calcofluor white staining and confocal Raman spectroscopy analysis revealed regular changes in the chemical composition of cell walls, such as relative lower cellulose deposition in transition stage in vascular cambium, and higher lignin accumulation was found in dormant stage in secondary xylem. Moreover, real-time quantitative polymerase chain reaction analysis suggested that various IAA (Aux/IAA protein), CesA, CslA and HDZ genes, as well as NAC, PME3 and PME4, may be involved in cambium activities and secondary xylem formation. Taken together, these findings provide new information about cambium activity and cell differentiation in the formation, structure and chemistry in conifers during the active-dormant transition.

Analyses of Microstructure and Dynamic Deposition of Cell Wall Components in Xylem Provide Insights into Differences between Two Black Poplar Cultivars

The chemical composition of the cell wall varies between species and even within the same species, and impacts the properties of the cell wall. In this study, the dynamic chemical compositions of the xylem cell walls of two black poplar cultivars, Populus × euramericana ‘Zhonglin46’ and Populus × euramericana ‘Neva,’ were investigated in situ using stimulated Raman scattering microscopy (SRS). Meanwhile, the pectin structural features were examined using immunofluorescence methods. The results showed that Neva displayed faster thickening of the fiber cell walls than Zhonglin46 did, and it had a greater cell wall thickness in mature xylem. A faster deposition speed of lignin and cellulose during xylem maturation was revealed in Neva. Significantly higher lignin contents were found in the mature xylem of Neva compared with those of Zhonglin46, while no obvious differences in cellulose deposition in mature xylem were observed between the two cultivars. The patterns of pectin deposition during xylem maturation were similar in the two cultivars, but more pectin was found in the mature xylem of Neva than in that of Zhonglin46. The chemical deposition patterns account for the anatomical feature differences between the cultivars. These results provide valuable insights into the chemical deposition and anatomical differences between cultivars, and they might be helpful in understanding the wood growth processes and facilitating the utilization of different poplar cultivars.

Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) spectra of salivary glands carcinoma, tumor and healthy tissues and their homogenates analyzed by chemometry: Towards development of the novel tool for clinical diagnosis

In this study, two approaches to salivary glands studies are presented: Raman imaging (RI) of tissue cross-section and surface-enhanced Raman spectroscopy (SERS) of tissue homogenates prepared according to elaborated protocol. Collected and analyzed data demonstrate the significant potential of SERS combined with multivariate analysis for distinguishing carcinoma or tumor from the normal salivary gland tissues as a rapid, label-free tool in cancer detection in oncological diagnostics. Raman imaging allows a detailed analysis of the cell wall's chemical composition; thus, the compound's distribution can be semi-quantitatively analyzed, while SERS of tissue homogenates allow for detailed analysis of all moieties forming these tissues. In this sense, SERS is more sensitive and reliable to study any changes in the area of infected tissues. Principal component analysis (PCA), as an unsupervised pattern recognition method, was used to identify the differences in the SERS salivary glands homogenates. The partial least squares-discriminant analysis (PLS-DA), the supervised pattern classification technique, was also used to strengthen further the computed model based on the latent variables in the SERS spectra. Moreover, the chemometric quantification of obtained data was analyzed using principal component regression (PCR) multivariate calibration. The presented data prove that the PCA algorithm allows for 91% in seven following components and the determination between healthy and tumor salivary gland homogenates. The PCR and PLS-DA methods predict 90% and 95% of the variance between the studied groups (in 6 components and 4 factors, respectively). Moreover, according to calculated RMSEC (RMSEP), R2C (R2P) values and correlation accuracy (based on the ROC curve), the PLS-DA model fits better for the studied data. Thus, SERS methods combined with PLS-DA analysis can be used to differentiate healthy, neoplastic, and mixed tissues as a competitive tool in relation to the commonly used method of histopathological staining of tumor tissue.

Advanced mycelium materials as potential self-growing biomedical scaffolds

Mycelia, the vegetative part of fungi, are emerging as the avant-garde generation of natural, sustainable, and biodegradable materials for a wide range of applications. They are constituted of a self-growing and interconnected fibrous network of elongated cells, and their chemical and physical properties can be adjusted depending on the conditions of growth and the substrate they are fed upon. So far, only extracts and derivatives from mycelia have been evaluated and tested for biomedical applications. In this study, the entire fibrous structures of mycelia of the edible fungi Pleurotus ostreatus and Ganoderma lucidum are presented as self-growing bio-composites that mimic the extracellular matrix of human body tissues, ideal as tissue engineering bio-scaffolds. To this purpose, the two mycelial strains are inactivated by autoclaving after growth, and their morphology, cell wall chemical composition, and hydrodynamical and mechanical features are studied. Finally, their biocompatibility and direct interaction with primary human dermal fibroblasts are investigated. The findings demonstrate the potentiality of mycelia as all-natural and low-cost bio-scaffolds, alternative to the tissue engineering systems currently in place.

Effects of natural weathering on the chemical composition of cell walls in sapwood and heartwood of Japanese cedar

Effects of natural weathering on the chemical composition of cell walls in sapwood and heartwood of Japanese cedar

A label-free, fast and high-specificity technique for plant cell wall imaging and composition analysis

BackgroundNew cell wall imaging tools permit direct visualization of the molecular architecture of cell walls and provide detailed chemical information on wall polymers, which will aid efforts to use these polymers in multiple applications; however, detailed imaging and quantification of the native composition and architecture in the cell wall remains challenging.ResultsHere, we describe a label-free imaging technology, coherent Raman scattering (CRS) microscopy, including coherent anti-Stokes Raman scattering (CARS) microscopy and stimulated Raman scattering (SRS) microscopy, which can be used to visualize the major structures and chemical composition of plant cell walls. We outline the major steps of the procedure, including sample preparation, setting the mapping parameters, analysis of spectral data, and image generation. Applying this rapid approach will help researchers understand the highly heterogeneous structures and organization of plant cell walls.ConclusionsThis method can potentially be incorporated into label-free microanalyses of plant cell wall chemical composition based on the in situ vibrations of molecules.

Cell wall epitopes in grasses of different novel ecosystem habitats on post‐industrial sites

The post-industrial habitats provide previously unknown conditions for plant and vegetation development. We asked the question: do the extreme soil substrate conditions cause differences in chemical composition of cell walls of leaves in three grass species: Calamagrostis epigejos (L.) Roth, Phragmites australis (Cav.) Trin. ex Steud, and Molinia caerulea (L.) Moench? With the use of immunohistochemical methods we determined the spatial distribution of selected pectic and AGP (arabinogalactan proteins) epitopes within leaf tissues of grass species growing in two industrial and control (meadow) habitats. Some post-industrial habitats increase biodiversity. Fast adaptation and divergence cause phenotypic changes. This process has been recently recorded and is called human-induced rapid evolutionary changes. The levels of pectins and arabinogalactan proteins increased on the post-industrial habitats in comparison to the control sites. On control habitat, pectic epitope with galactan chain residues (recognised by the LM5 antibody) was not detected in cell walls of Calamagrostis epigejos leaves, but was abundantly present in Phragmites australis leaves. The pectic epitope with arabinan residues (recognized by LM6 antibody) was less represented in cell walls of Molinia caerulea leaves. AGP epitope, recognised by LM2 antibody, was abundant only in Ph. australis. In Molinia caerulea this epitope was not detected. The AGP epitope recognised by JIM13 antibody was observed in all analysed species. The results obtained revealed the response of grass individuals to different environments: a) varied responses to different soil substratum conditions (novel ecosystems), and b) that this reaction is species-specific with respect to the analysed cell wall components.

Microanalytical techniques for phenotyping secondary xylem

Abstract The products of secondary xylem are of significant biological and commercial importance, and as a result, the biology of secondary growth and how intrinsic and extrinsic factors influence this process have been the subject of intense investigation. Studies into secondary xylem range in scale from the cellular to the forest stand level, with phenotypic analyses often involving the assessment of traits relating to cell morphology and cell wall chemical composition. While numerous techniques are currently available for phenotypic analyses of samples containing abundant amounts of secondary tissue, only a few of them (microanalytical techniques) are suitable when working with limiting amounts of secondary tissue or where a fine-scale resolution of morphological features or cell wall chemical composition is required. While polarised light microscopy, scanning electron microscopy, field emission-scanning electron microscopy and X-ray scattering and micro-tomography techniques serve as the most frequently used microanalytical techniques in morphotyping, techniques such as scanning ultraviolet microspectrophotometry, X-ray photoelectron spectroscopy, gas chromatography, Fourier-transform infrared spectroscopy and matrix-assisted laser desorption ionisation mass spectrometry serve as the most commonly used microanalytical techniques in chemotyping. Light microscopy, fluorescence microscopy, confocal laser scanning microscopy, transmission electron microscopy and Raman spectroscopy serve as dual micro morphotyping and chemotyping techniques. In this review, we summarise and discuss these techniques in the light of their applicability as microanalytical techniques to study secondary xylem.

The class II KNOX transcription factors KNAT3 and KNAT7 synergistically regulate monolignol biosynthesis in Arabidopsis.

The function of the transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) is still unclear since it appears to be either a negative or a positive regulator for secondary cell wall deposition with its loss-of-function mutant displaying thicker interfascicular and xylary fiber cell walls but thinner vessel cell walls in inflorescence stems. To explore the exact function of KNAT7, class II KNOTTED1-LIKE HOMEOBOX (KNOX II) genes in Arabidopsis including KNAT3, KNAT4, and KNAT5 were studied together. By chimeric repressor technology, we found that both KNAT3 and KNAT7 repressors exhibited a similar dwarf phenotype. Both KNAT3 and KNAT7 genes were expressed in the inflorescence stems and the knat3 knat7 double mutant exhibited a dwarf phenotype similar to the repressor lines. A stem cross-section of knat3 knat7 displayed an enhanced irregular xylem phenotype as compared with the single mutants, and its cell wall thickness in xylem vessels and interfascicular fibers was significantly reduced. Analysis of cell wall chemical composition revealed that syringyl lignin was significantly decreased while guaiacyl lignin was increased in the knat3 knat7 double mutant. Coincidently, the knat3 knat7 transcriptome showed that most lignin pathway genes were activated, whereas the syringyl lignin-related gene Ferulate 5-Hydroxylase (F5H) was down-regulated. Protein interaction analysis revealed that KNAT3 and KNAT7 can form a heterodimer, and KNAT3, but not KNAT7, can interact with the key secondary cell wall formation transcription factors NST1/2, which suggests that the KNAT3-NST1/2 heterodimer complex regulates F5H to promote syringyl lignin synthesis. These results indicate that KNAT3 and KNAT7 synergistically work together to promote secondary cell wall biosynthesis.