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We studied the time-course of stem response for six months following complete girdling in branches of Aquilaria sinensis to determine the potential role of interxylary phloem (IP) in this response. It was found that the vascular cambium, as well as its derivative secondary xylem and phloem, regenerated fully through redifferentiation of IP. We confirmed that vascular cambium regenerated within one month after girdling based on observation of new vessels, IP, and secondary phloem fibers. The time-course study showed that IPs made connections with each other, merged, and became larger through the proliferation of IPs parenchyma cells and the cleaving of secondary xylem in a narrow zone 400 to 1000 μm deep inside the girdled edge. This led to the formation of a complete circular sheath of vascular cambium, followed by the regeneration of vascular tissue. It is worth noting that the secondary xylem is regenerated always following the formation of a thick belt of wound xylem.

The feruloylarabinoxylan deposition was initiated at the formation of the secondary cell wall, especially S

Xylan deposition and lignification processes were examined in tension wood fibers with gelatinous layers (G-layers) in Mallotus japonicus (Euphorbiaceae). The cell walls consisted of a multi-layered structure of S1 + S2 + G + n(L + G), where n indicates the number of repetitions (n = 0–3) and L indicates very thin lignified layers. The formation and lignification processes of the multi-layered structure of tension wood fibers were examined by light microscopy, ultraviolet microscopy, and transmission electron microscopy (TEM) following KMnO4 staining. The deposition of xylan was examined by immunoelectron microscopy with a monoclonal antibody (LM11). Immunolabelling of xylan appeared in lignified cell wall layers, except in the compound middle lamella (CML), i.e., the S1, S2, and L layers but not the G-layers. The density of LM11 xylan immunogold labeling in S2 layers increased during the formation of G-layers. This increase was due to the shrinkage of S2 layers during development rather than intrusive deposition of xylan through G-layers. Lignification of the CML, S1, and S2 layers proceeded during G-layer formation. The shrinkage of S2 layers occurred almost simultaneously with the lignification of the S2 layers during G-layer formation, suggesting that the S2 layers shrank with lignification.

Oligomers of β-amyloid 42 (Aβ42), rather than fibrils, drive the pathogenesis of Alzheimer's disease (AD). In particular, toxic oligomeric species called protofibrils (PFs) have attracted significant attention. Herein, we report RNA aptamers with higher affinity toward PFs derived from a toxic Aβ42 dimer than toward fibrils produced from WT Aβ42 or from a toxic, conformationally constrained Aβ42 variant, E22P–Aβ42. We obtained these RNA aptamers by using the preincubated dimer model of E22P–Aβ42, which dimerized via a linker located at Val-40, as the target of in vitro selection. This dimer formed PFs during incubation. Several physicochemical characteristics of an identified aptamer, E22P–AbD43, suggested that preferential affinity of this aptamer toward PFs is due to its higher affinity for the toxic dimer unit (KD = 20 ± 6.0 nm) of Aβ42 than for less-toxic Aβ40 aggregates. Comparison of CD data from the full-length and random regions of E22P–AbD43 suggested that the preferential binding of E22P–AbD43 toward the dimer might be related to the formation of a G-quadruplex structure. E22P–AbD43 significantly inhibited the nucleation phase of the dimer and its associated neurotoxicity in SH-SY5Y human neuroblastoma cells. Of note, E22P–AbD43 also significantly protected against the neurotoxicity of WT Aβ42 and E22P–Aβ42. Furthermore, in an AD mouse model, E22P–AbD43 preferentially recognized diffuse aggregates, which likely originated from PFs or higher-order oligomers with curvilinear structures, compared with senile plaques formed from fibrils. We conclude that the E22P–AbD43 aptamer is a promising research and diagnostic tool for further studies of AD etiology.

Summary The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression‐wood‐specific S2L cell wall layer with lignins highly enriched with p‐hydroxyphenyl (H)‐type units derived from H‐type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)‐type units from G‐type monolignol with a trace amount of H‐type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H‐type and G‐type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa).We performed a series of chemical‐probe‐aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases.Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H‐type and G‐type monolignols were precisely localised to distinct cell wall layers in which H‐type and G‐type lignin units were preferentially produced during the development of compression wood tracheids.We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

In the original publication of the article, the letter “l” in the section headings was published incorrectly as “l”. It should appear as “l” throughout the article.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly. Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-l-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II

Suspensions of cellulose nanocrystals (CNCs) having different allomorphs were prepared from pulp and microcrystalline cellulose. Comparison found varying particle features. In situ observation of cellulose enzymes interacting with CNC I, II and III by atomic force microscopy (AFM) and transmission electron microscopy (TEM) found diverse adsorption of the enzymes on the CNC. Greater degradation to glucose was revealed for short rod-like CNC II and thin needle-like CNC III than CNC I, which were mainly due to their greater d-spacings for the (110) (CNC II) and (1–10) (CNC III) planes compared to the (200) spacing for CNC I. According to the model crystal structures, the surface areas for those planes where the enzymes would adsorb were also lager for CNC II and III. However, CNC III had significantly enhanced adsorption capability compared to the low affinity of CNC II. Enzymes tended to gather near hydrophobic surfaces that correspond to planes with small crystallite sizes on CNC III, and the enzymes caused breaks of nanocrystals during the degradation. Understanding the interactions between CNC allomorphs and cellulase would provide deep insights into biodegradation of crystalline cellulose as well as extensive application of CNCs.

A gradual shift in the microfibril angle of gelatinous layer (G-layer) of tension wood fibres of the S1+G type has been detected via potassium permanganate (KMnO4) staining. Thus, microfibril angles in fibres of the S1+G type are different from S1+S2+G type fibres. We evaluated the microfibril orientation and presence of lignin and xylan in G-layers of tension wood fibres of the S1+G type in several Japanese hardwoods. The distribution of xylan and lignin was examined using immunoelectron microscopy with anti-xylan monoclonal antibody, ultraviolet (UV) microscopy, fluorescence microscopy after acrifravine staining and transmission electron microscopy after KMnO4 staining. In transverse sections, the outer parts of the G-layers showed ultraviolet absorption and a heterogeneous KMnO4 staining pattern, suggesting that lignin was heterogeneously distributed in the outer parts of the G-layers. The heterogeneous staining pattern was found in the G-layers of several tree species; however, the degree of staining differed between tree species. In longitudinal sections, the KMnO4-staining region in the G-layers continued parallel to the cell axis to variable lengths. The orientation of cellulose microfibrils changed gradually from a steep helix to parallel to the cell axis from the outer to inner parts of the G-layers. Xylan immunolabelling was observed in the outer part of the G-layers; in some fibres, labelling was found in the innermost parts of the G-layers. Following immunogold labelling combined with KMnO4 staining, xylan labelling was mainly found in KMnO4-stained electron-opaque regions, suggesting that lignin and xylan were heterogeneously colocalized in the outer parts of the G-layers. The rotation of cellulose microfibrils and heterogeneous distribution of xylan and lignin might be a general phenomenon in S1+G tension wood fibres.

Ionic liquid (IL) is one of the pretreatment processes gaining considerable interests to remove the native recalcitrance of lignocellulose. But the cellulose crystalline transformation during the pretreatment and their correlations with enzymatic digestibility have not been fully elucidated. Microcrystalline cellulose (Avicel) and holocellulose, which have differential sources and original crystallinity, were respectively pretreated with 1-butyl-3-methylimidazolium chloride ([C4min]Cl). Cellulose crystalline variations as well as chemical and morphological changes were determined. Crystallinity of different materials was proved to influence the effects of pretreatment and following enzymatic digestibility. Recrystallized cellulose Iβ was revealed from slight initial cellulose Iα of Avicel, which was accomplished via formation of intermediate paracrystalline phases. The conversion yield of IL pretreated Avicel displayed no obvious changes, mainly resulted from initial high crystalline order and the recrystallization behavior. Recalcitrance of holocellulose was destroyed during cellulose allomorph transformation and hemicelluloses extraction, contributing to significant increase of glucose yield up to 92.20%. Explicit comprehension on cellulose supramolecular structure may help provide more efficient process for bioconversion after IL pretreatment.

Wood rotting basidiomycetes produce extracellular mucilaginous sheaths interfacing fungal hyphae and plant biomass. While the versatility of these fungal sheaths has been addressed, sheaths generated by selective white-rot fungi remain poorly understood. To fill this gap, the sheath produced by the basidiomycete Ceriporiopsis subvermispora, which degrades lignin while inflicting limited cellulose damage, was analyzed in this study. Fluorescence and transmission electron microscopy revealed that the sheath formed three days after inoculation into a beech wood slice on an agar plate and was embedded at the interface between fungal hyphae and wood cell walls. The sheath's chemical structure was evaluated from fungus cultures in a liquid medium containing [U-13C6]-d-glucose and beech wood slices. Compositional analysis, methylation analysis, and 13C NMR demonstrated that the sheath mainly consisted of a comb-like β-1,6-glucopyranose residue-branched β-1,3-glucan, which is advantageous to retain water and extracellular secondary metabolites.

l-Coniferin (1L), which is an enantiomer of natural coniferin (d-coniferin (1D)), was prepared from l-glucose according to the conventional method for compound 1D. The reactivity of l-glucose and its derivatives was found to be almost same as that of the corresponding d-glucose and its derivatives during the preparation for compound 1L. Compound 1L showed resistance toward enzymatic hydrolysis by commercial β-glucosidase from Almond. However, unlike compound 1D, compound 1L was not transported across the membrane obtained from differentiating xylem of a hybrid poplar in the present assay. The result suggested for the first time that the d-/l-configuration of the glucose moiety of coniferin is an important factor affecting coniferin transport across the membrane.

Lignin is a cell wall component of vascular plants crucial for survival in terrestrial environments. While p-hydroxyphenyl lignin is minor, it is considered to be localised in the outermost part of the cell wall providing strong adhesion between cells, which determines cell shape. Transport of the lignin precursor from the cytosol to the cell wall is critical to regulate temporal and spatial lignin deposition; however, little information on the transport step is available. Here, we report transport activity of p-glucocoumaryl alcohol, a precursor of p-hydroxyphenyl lignin, in a broad-leaved tree (hybrid poplar, Populus sieboldii × P. grandidentata) and a coniferous tree (Japanese cypress, Chamaecyparis obtusa). Membrane vesicles of both trees were prepared from differentiating xylem with vigorous lignification and used for transport assays. Several inhibition assays indicated that not ABC transporters but the proton gradient and V-ATPase are involved in p-glucocoumaryl alcohol transport depending on ATP. These results support the hypothesis that p-glucocoumaryl alcohol is loaded into the secretory vesicles and delivered to the cell wall by exocytosis. Furthermore, this transport mechanism was common in both poplar and Japanese cypress, strongly suggesting that p-glucocoumaryl alcohol transport in the differentiating xylem is conserved within woody plants.

Pretreatment is an essential step to effectively hydrolyze lignocellulosic polysaccharides. In this paper, we investigated the degree of decompositions of lignin and cell wall structure using dilute acid, alkali, and water pretreatments to assess both chemical and ultrastructural alterations during pretreatment. The thioacidolysis method showed that β-O-4 linkages in lignin were mostly cleaved after all pretreatments, in which the highest decrease of β-O-4 units was for NaOH pretreatment, followed by hot water and H2SO4 pretreatments. The amounts of lignin degradation compounds, including vanillin and syringaldehyde, in the supernatant water also differed between the three pretreatments. Field-emission scanning electron microscopy revealed clear differences among the pretreatments in decomposing the ultrastructure of the inner surface of the fiber cell walls. Small pores were formed due to degradation of a part of the warty layer of the innermost surface in H2SO4 pretreatment. The warty layer was more degraded in hot water pretreatment and thus the cellulose microfibrils of the secondary walls were exposed. NaOH pretreatment showed that the warty layer was almost completely decomposed. The comparative study of different pretreatments using chemical methods and microscopic observations led to a better understanding of decomposition of wood cell walls by thermochemical pretreatment.

The traditional Maule color reaction was improved by use of tris(hydroxymethyl) aminomethane-hydrochloride acid (Tris–HCl) buffer coupled with a fluorescence microscope. The purple-red color of Maule-treated hardwood was more stable with the novel treatment than with traditional treatment. In addition, Maule-treated wood samples had a characteristic fluorescence when excited with blue (460–495 nm) light. Examination of white birch (Betula platyphylla) showed that syringyl-rich and guaiacyl-rich cell walls emitted red and yellow fluorescence, respectively. Measuring the fluorescence spectra of hardwood and softwood powders after treatment with the new Maule reagents showed that hardwood powder containing syringyl lignin emitted a red fluorescence, whereas softwood powder containing guaiacyl lignin emitted a green fluorescence when excited with blue light. In conclusion, this improved technique has many advantages compared with the traditional Maule color test.

A comprehensive understanding of the structure and properties of gramineous lignocelluloses is needed to facilitate their uses in biorefinery. In this study, lignocelluloses from fractionated internode tissues of two taxonomically close species, Erianthus arundinaceus and sugarcane (Saccharum spp.), were characterized. Our analyses determined that syringyl (S) lignins were predominant over guaiacyl (G) or p-hydroxyphenyl (H) lignins in sugarcane tissues; on the other hand, S lignin levels were similar to those of G lignin in Erianthus tissues. In addition, tricin units were detected in sugarcane tissues, but not in Erianthus tissues. Distributions of lignin inter-monomeric linkage types were also different in Erianthus and sugarcane tissues. Alkaline treatment removed lignins from sugarcane tissues more efficiently than Erianthus tissues, resulting in a higher enzymatic digestibility of sugarcane tissues compared with Erianthus tissues. Our data indicate that Erianthus biomass displayed resistance to alkaline delignification and enzymatic digestion.

As the main hemicellulose of poplar, the interaction of xylan with lignin was expected to have profound effect on biomass recalcitrance. In this paper, the dynamic changes of xylan and lignin in poplar cell wall during a mild pretreatment using γ-valerolactone (GVL) was investigated using chemical and microscopic techniques. Synergetic dissolution of branched xylan and lignin from the secondary wall of the fiber cell was found to play a major role in opening the cell wall structure for enzymatic attack. In the case of the removal of xylan and lignin reaching a certain level, β-O-4′ cleavage of lignin which destroyed its interaction with hydrophobic cellulose face was found to make great contribution to the enhanced enzymatic hydrolysis. The deep understanding of this process could lead to a new insight into the understanding of the plant cell wall architecture and provide basic information for biomass processing.

The distribution of lignin, 8-5′ and 8-8′ linked lignin substructure, and noncellulosic polysaccharides in hemp (Cannabis sativa L.) phloem fibers were explored based on histochemical and immunological methods. Ultraviolet absorption and potassium permanganate staining were observed mainly in the compound middle lamella (CML) and S1 layers, and rarely in the G-layer of phloem fibers, suggesting that lignin concentration is high at the CML and S1 layers, and very low at the G-layer of hemp fibers. Acriflavine staining, uniform KM1 labeling (8-5′ linked lignin substructure), and no KM2 labeling (8-8′ linked structure) were observed in the G-layer, suggesting that there is a small amount of lignin-like compound with 8-5′ linked structure in the G-layer. In addition, some fiber cells showed a multilayered structure. Uniform arabinogalactan protein (AGP) labeling was observed on the S1 layers and G-layers using JIM14, but little appeared in the CML of hemp fibers, indicating that these layers of the phloem fibers contain AGP. Immunogold labeling of xylan (LM11) and glucomannan (LM21) showed that xylan and glucomannan were mainly present in the S1 layers and the G-layers, respectively. In some phloem fibers, LM21 immunofluorescence labeling showed multilayered structure, suggesting the heterogeneous distribution of glucomannan.

Lignin is crucial for the growth and persistence of vascular plants in diverse environments. Although biosynthesis and polymerization of lignin precursors have been subjected to in-depth studies, only limited information exists on the ways in which these precursors are transported. Recent research has demonstrated that V-ATPase-dependent transport of coniferyl alcohol glucoside (coniferin) is a major metabolic function in differentiating xylem of both angiosperms and gymnosperms. Localization of β-glucosidase involved in the cleavage of coniferin to coniferyl alcohol and glucose is important to clarify the role of coniferin in lignification; nevertheless, only little has been reported in angiosperms. Here, we demonstrated the presence of coniferin β-glucosidase activity in a cell wall ionically bound protein fraction extracted from differentiating xylem of poplar. Coniferin β-glucosidase localization is very likely in cell wall similar to previous reports in conifers. We identified a putative poplar coniferin β-glucosidase through phylogenetic analysis and named this protein “PtrBGL6”. Immunoprecipitation assays showed that the anti-PtrBGL6 antibody recognizes a coniferin β-glucosidase in a cell wall ionically bound protein fraction. Conserved coniferin β-glucosidases localized in cell wall in both angiosperm and gymnosperm implies their important roles in the formation of lignified cell wall.

This study reports a method that combines near-infrared (NIR) measurements with multivariate analysis to predict the saccharification efficiency of hydrothermally pretreated Eucalyptus globulus during ethanol conversion. Optimization of the NIR data with or without spectral treatment determined the best calibration model in the region 10000–4000 cm −1 of the original spectra, with an RMSEP of 2.08% and R p 2 of 0.99. By investigating the regression coefficient to understand the key regions and chemical components, for original and multiplicative scatter correction (MSC)-treated spectra, the water absorption and higher wavenumber regions were important. For the second derivative spectra, the regression model was constructed based on the CH overtone vibrations (6050–5500 cm −1 ). The regression coefficient demonstrated that the removal of hemicellulose resulted in higher lignin content, which might affect the biomass properties in terms of water absorption and enhanced enzymatic hydrolysis evaluated by dinitrosalicylic acid (DNS) method. For a higher throughput system, aqueous sample analysis was performed using an immersion probe equipped with an InGaAs detector, which generated an acceptable calibration model having RMSEP of 4.25% and R p 2 of 0.94. These results show the great potential of NIR spectroscopy for achieving fast, accurate, and nondestructive analysis, and its highly adaptability for maintaining an ethanol bioconversion system.TGS, triglycine sulfate

To elucidate the influence of wood polysaccharide components on lignin formation in vitro, models for polysaccharide matrix in wood secondary cell wall were fabricated from two types of bacterial cellulosic films, flat film (FBC) and honeycomb-patterned film (HPBC), as basic frameworks by depositing xylan onto the films. An endwise type of dehydrogenative polymerization, "Zutropfverfahren", of coniferyl alcohol was attempted in the films with/without xylan. The resultant dehydrogenation polymer (DHP) was generated inside and outside xylan-deposited films, whereas DHP was deposited only outside the films without xylan. The amount of the generated DHP in the xylan-deposited films was larger than that in the films without xylan. The frequency of 8-O-4' interunitary linkage in DHP was also increased by the xylan deposition. These results suggest that xylan acts as a scaffold for DHP deposition in polysaccharides matrix and as a structure regulator for the formation of the 8-O-4' linkage. In addition, mechanical properties, i.e., tensile strength and modulus of elasticity (MOE), of both cellulosic films were found to be augmented by the deposition of xylan and DHP. Especially, DHP deposition remarkably enhanced MOE. Such effects of xylan on DHP formation and augmentation of mechanical strength were clearly observed for HPBC, revealing that HPBC is a promising framework model to investigate wood cell wall formation in vitro.

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. The RG-II region serves as the site of borate cross-linking within pectin, via which pectin macromolecules link together to form a gel. In this study, we examined whether RG-II is present in the cell plate, the precursor of primary cell walls that forms during cytokinesis. A structure inside dividing cells was labeled with a rabbit polyclonal anti-RG-II antibody and detected by immunofluorescence microscopy. An antibody against callose, a marker polysaccharide for the cell plate, also labeled the structure. In immunoelectron microscopy analyses using the anti-RG-II antibody, gold particles were distributed in electron-lucent vesicular structures that appeared to correspond to the forming cell plates in late anaphase cells. Together, these results suggest that RG-II is present in cell plates from the early phase of their assembly.

Introduction Hesperidin, a flavonoid known to have important pharmacological effects, accumulates particularly in the peels of satsuma mandarin (Citrus unshiu). Although histochemical studies have suggested that hesperidin forms crystals in some tissues of the Rutaceae and Umbelliferae, there has been no rigorous in situ detection or identification of hesperidin crystals in C. unshiu. Objective To characterise the chemical component of the crystals found in C. unshiu peels using Raman microscopy. Methods Sections of C. unshiu peels were made. The distribution and morphology of crystals in the sections were analysed microscopically. Raman microscopy was used to detect hesperidin in the sections directly. Results The crystals were more abundant in immature peel and were observed particularly in areas surrounding vascular bundles, around the border between the flavedo and albedo layers and just below the epidermal cells. In the morphological analysis by scanning electron microscopy, needle-shaped crystals aggregated and formed clusters of spherical crystals. Spectra obtained by Raman microscopy of the crystals in the peel sections were consistent with those of the hesperidin standard. Conclusion This study showed the detailed distribution of crystals in C. unshiu peels and their main component was identified using Raman microscopy to be hesperidin for the first time. Copyright © 2014 John Wiley & Sons, Ltd.

Immunolabeling by using monoclonal antibodies showed that xylan deposition precedes the formation of 8-5′-linked structure of lignin in normal and compression woods of Chamaecyparis obtusa. Xylan deposition and formation of 8-5′-linked lignin structure in differentiating xylems from normal and compression woods in Chamaecyparis obtusa were examined by immunoelectron microscopy using monoclonal antibodies (LM10 or LM11) to detect xylan localization. The 8-5′-linked lignin structure was immunolocalized using KM1 antibody. Xylan and 8-5′-linked lignin double immunolabeling was performed using secondary antibodies labeled with colloidal gold particles of different diameters. In normal wood, KM1 labeling occurred in the compound middle lamella (CML) and S1 layer during S1 layer formation and increased as S2 and S3 layers formed, with labeling occurring at the outer part of the previous layer. In compression wood, mild KM1 labeling occurred in the CML and outer part of the S1 layer at the later S1 layer formation stage, with increased labeling as the S2 layer formed. Minor labeling occurred in the outer part of the S2 layer during helical cavity formation. Comparison between KM1 labeling and KMnO4 staining suggested that lignin other than 8-5′-linked structure was formed during early lignification, and the proportion of 8-5′-linked lignin structure increased at later stages of lignification in both normal and compression woods. LM10 and LM11 labeling occurred slightly earlier than KM1 labeling, suggesting that xylan deposition preceded the formation of 8-5′-linked lignin in normal and compression woods. Less labeling by KM1, LM10, and LM11 occurred in the outer part of the S2 layer in compression wood, which has abundant lignin. Thus, lignin in these parts is composed of lignin substructures other than the 8-5′ linkage.

Phloem fibres in Mallotus japonicus Müll. Arg. were found to have a multi-layered structure that is S1 + S2 + n(G + L), where a non-lignified gelatinous layer (G) and a lignified layer (L) are formed alternately and n indicates the number of repetitions of these two layers. The aim of this study was to determine the process of xylan deposition and lignification in the multi-layered cell walls of phloem fibres. The formation process of the multi-layered structure of secondary phloem fibres was examined by light microscopy, ultraviolet microscopy and transmission electron microscopy. The distribution of glucuronoxylan was examined by immunoelectron microscopy. The activity of peroxidase was also determined using metal-enhanced diaminobenzidine substrates. Immunolabelling of glucuronoxylan occurred in lignified cell wall layers, except in the compound middle lamella (CML), i.e., the S1, S2 and L layers but not the G layers. Change in immunolabelling density suggests that xylan deposition in these lignified layers occurs appositionally, i.e., xylan is deposited into the lignified layers directly and not by a penetrative mechanism, and deposition does not occur after the layers are fully deposited. Peroxidase activity was found in CML including cell corners during S2 layer formation, then in developing G layers during G layer formation. Peroxidase activity was also found in the thin L layers that formed recently and was not found in the L layers already present. Xylan labelling was not found in the thin L layers that formed recently but did occur in L layers that developed earlier. Lignification of the S1 and S2 layers continued during the formation of the G layers, whereas in the L layers it finished just after deposition of the L layer.

Lignin is an integral component of the cell wall of vascular plants. The mechanism of supply of lignin precursors from the cytosol into the cell wall of differentiating xylem has not yet been elucidated. The present study showed that a certain amount of coniferyl alcohol glucoside (coniferin) occurred in the differentiating xylem of Japanese cypress (Chamaecyparis obtusa), as previously reported in gymnosperms. Coniferin content peaked in the early stages of secondary wall formation and decreased during lignification. In contrast to gymnosperms, coniferin content was limited in the differentiating xylem of poplar (Populus sieboldii × Populus grandidentata). Moreover, coniferyl alcohol was not detected in all specimens. In the differentiating xylem of poplar, a higher amount of sinapyl alcohol occurred than glucoside (syringin). However, the phloem contained syringin and not sinapyl alcohol. The sinapyl alcohol content in the xylem peaked in the cells with ceasing cell wall formation, and decreased gradually towards the boundary of the annual ring, where the lignin content kept increasing. Sinapyl alcohol in the differentiating xylem of poplar may be used for the lignification of the xylem.

Lignin biosynthesis is an essential physiological activity of vascular plants if they are to survive under various environmental stresses on land. The biosynthesis of lignin proceeds in the cell wall by polymerization of precursors; the initial step of lignin polymerization is the transportation of lignin monomers from the cytosol to the cell wall, which is critical for lignin formation. There has been much debate on the transported form of the lignin precursor, either as free monolignols or their glucosides. In this study, we performed biochemical analyses to characterize the membrane transport mechanism of lignin precursors using angiosperms, hybrid poplar (Populus sieboldii × Populus grandidentata) and poplar (Populus sieboldii), as well gymnosperms, Japanese cypress (Chamaecyparis obtusa) and pine (Pinus densiflora). Membrane vesicles prepared from differentiating xylem tissues showed clear ATP-dependent transport activity of coniferin, whereas less than 4% of the coniferin transport activity was seen for coniferyl alcohol. Bafilomycin A1 and proton gradient erasers markedly inhibited coniferin transport in hybrid poplar membrane vesicles; in contrast, vanadate had no effect. Cis-inhibition experiments suggested that this transport activity was specific for coniferin. Membrane fractionation of hybrid poplar microsomes demonstrated that transport activity was localized to the tonoplast- and endomembrane-rich fraction. Differentiating xylem of Japanese cypress exhibited almost identical transport properties, suggesting the involvement of a common endomembrane-associated proton/coniferin antiport mechanism in the lignifying tissues of woody plants, both angiosperms and gymnosperms.

Cryofixation and freeze-substitution techniques preserve plant ultrastructure much better than conventional chemical fixation techniques. The advantage of cryofixation is not only in structural preservation, as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron microscopy of post-embedded, cryofixed plant tissues by applying an antibody to a thin plastic resin-embedded section prepared by cryofixation followed by freeze-substitution.

The rapid freeze and freeze substitution method for fixing the differentiating xylem has given us new information on cell wall assembly. Hemicelluloses are synthesized in the Golgi apparatus and transported to the inner surface of the developing cell wall by exocytosis of Golgi vesicles. As a result of hemicelluloses deposition, the inner surface of the developing cell walls is always coated with a thin layer composed of hemicelluloses and a significant amount of water. Lignification excludes water from nonlignified and lignifying cell walls. Immunocytochemistry reveals not only the localization of enzymes involved in the biosynthesis of cell wall components, but also the localization of hemicelluloses and lignins within the cell wall. Biochemical investigations suggest that the mechanism of monolignols transportation from the cell to the developing cell walls is by passing through the plasma membrane.

The lignification process in poplar tension wood lignified cell wall layers, specifically the S(1) and S(2) layers and the compound middle lamella (CML), was analysed using ultraviolet (UV) and transmission electron microscopy (TEM). Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the lignified cell wall layers had completed their lignification before the deposition of G-layers, or, on the contrary, if lignification of these layers was still active during G-layer formation. Observations using UV microscopy and TEM indicated that both UV absorbance and the degree of potassium permanganate staining increased in the CML and S(1) and S(2) layers during G-layer formation, suggesting that the lignification of these lignified layers is still in progress during G-layer formation. In the context of the cell-autonomous monolignol synthesis hypothesis, our observations suggest that monolignols must go through the developing G-layer during the lignification of CML and the S(1) and S(2) layers. The alternative hypothesis of external synthesis (in the rays) does not require that monolignols go through the G-layer before being deposited in the CML, or the S(1) and S(2) layers. Interestingly, the previous observation of lignin in the poplar G-layer was not confirmed with the microscopy techniques used in the present study.

A novel anaerobic, Fe(III)-reducing, hydrogenogenic, carboxydotrophic bacterium, designated strain Ug1T, was isolated from a volcanic acidic hot spring in southern Kyushu Island, Japan. Cells of the isolate were rod-shaped (1.0–3.0 µm long) and motile due to peritrichous flagella. Strain Ug1Tgrew chemolithoautotrophically on CO (100 % in the gas phase) with reduction of ferric citrate, amorphous iron (III) oxide, 9,10-anthraquinone 2,6-disulfonate, thiosulfate or elemental sulfur. No carboxydotrophic growth occurred with sulfate, sulfite, nitrate or fumarate as electron acceptor. During growth on CO, H2and CO2were produced. Growth occurred on molecular hydrogen as an energy source and carbon dioxide as a sole carbon source. Growth was observed on various organic compounds under an N2atmosphere with the reduction of ferric iron. The temperature range for carboxydotrophic growth was 50–70 °C, with an optimum at 65 °C. The pH25 °Crange for growth was 4.6–8.6, with an optimum between 6.0 and 6.5. The doubling time under optimum conditions using CO with ferric citrate was 1.5 h. The DNA G+C content was 42.2 mol%. Analysis of 16S rRNA gene sequences demonstrated that this strain belongs to the thermophilic carboxydotrophic bacterial genusCarboxydothermus, with sequence similarities of 94.1–96.6 % to members of this genus. The isolate can be distinguished from other members of the genusCarboxydothermusby its ability to grow with elemental sulfur or thiosulfate coupled to CO oxidation. On the basis of phylogenetic analysis and unique physiological features, the isolate represents a novel species of the genusCarboxydothermusfor which the nameCarboxydothermus pertinaxsp. nov. is proposed; the type strain of the novel species is Ug1T( = DSM 23698T = NBRC 107576T).

The ultrastructure of the innermost surface of Cryptomeria japonica differentiating normal wood (NW) and compression wood (CW) was comparatively investigated by field emission electron microscopy (FE-SEM) combined with enzymatic degradation of hemicelluloses. Cellulose microfibril (CMF) bundles were readily observed in NW tracheids in the early stage of secondary cell wall formation, but not in CW tracheids because of the heavy accumulation of amorphous materials composed mainly of galactans and lignin. This result suggests that the ultrastructural deposition of cell wall components in the tracheid cell wall differ between NW and CW from the early stage of secondary cell wall formation. Delignified NW and CW tracheids showed similar structural changes during differentiating stages after xylanase or β-mannanase treatment, whereas they exhibited clear differences in ultrastructure in mature stages. Although thin CMF bundles were exposed in both delignified mature NW and CW tracheids by xylanase treatment, ultrastructural changes following β-mannanase treatment were only observed in CW tracheids. CW tracheids also showed different degradation patterns between xylanase and β-mannanase. CMF bundles showed a smooth surface in delignified mature CW tracheids treated with xylanase, whereas they had an uneven surface in delignified mature CW tracheids treated with β-mannanase, indicating that the uneven surface of CMF bundles was related to xylans. The present results suggest that ultrastructural deposition and organization of lignin and hemicelluloses in CW tracheids may differ from those of NW tracheids.

In this paper, we report the combination of a near-infrared (NIR) spectroscopic method with multivariate analysis in order to develop a calibration model of the saccharification ratio of chemically pretreated Erianthus. The regression models clearly depend on the NIR spectral regions, and the information of CH and aromatic framework vibrations contributed most effectively to the alkaline dataset. From interpretations of the regression coefficient, lignin and cellulose were negatively and positively correlated with the saccharification ratio, respectively, and this result was supported by the data from wet chemical analysis. A more complex dataset was obtained from varied chemical pretreatments; here, the saccharification ratio was either small or had no linear correlation with each structural monocomponent. These results enabled the successful construction of the PLS regression model. NIR spectroscopy can be a rapid screening method for the saccharification ratio, and furthermore, can provide information of the key factors influencing the realization of more efficient enzymatic accessibility.

The distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in warts and the warty layer of tracheids in normal wood (NW) and compression wood (CW) of Cryptomeria japonica was investigated. Under field emission scanning electron microscope (FE-SEM) observation, warts and the warty layer of delignified NW and CW tracheids were degraded by xylanase treatment, indicating that warts and the warty layer contain high amounts of AGXs. However, the effect of xylanase was not observed in NW and CW tracheids before delignification, suggesting that AGXs in warts and the warty layer may be encrusted with lignin. After β-mannanase treatment, no noticeable changes were observed in warts and the warty layer of NW tracheids, indicating that warts and the warty layer contain either no or very few GGMs. Similar results to FE-SEM observations were also observed with immunogold labeling. AGX labeling was observed in warts and the warty layer of NW and CW tracheids, while GGM labeling was not detected. NW tracheids showed a much stronger density of AGX labeling than did CW tracheids in warts and the warty layer, indicating differences in the chemical compositions of warts and the warty layer between NW and CW tracheids. Keywords :O-acetyl-galactoglucomannans (GGMs), arabino-4-O-methylglucuronoxylans (AGXs), compression wood, Cryptomeria japonica, wart, warty layer

A living wood fiber (LWF) is one that retains the living protoplast. LWFs store numerous starch grains during the dormant period. In black locust (Robinia pseudoacacia), almost all wood fibers in the outer part of the annual ring are LWFs. In the outermost ring, starch is accumulated during the summer, retained in winter, and metabolized during spring. We determined the starch content of LWFs, ray parenchyma, and axial parenchyma using image analysis. More than 70% of the starch grains in the outermost ring were stored in LWFs during winter. After the breakdown of starch in spring, LWFs resulted in cell death. These results indicate that LWFs in black locust function as “single-use” large-capacity starch storage.

Compression wood (CW) tracheids have different cell wall components than normal wood (NW) tracheids. However, temporal and spatial information on cell wall components in CW tracheids is poorly understood. We investigated the distribution of arabino-4-O-methylglucuronoxylans (AGXs) and O-acetyl-galactoglucomannans (GGMs) in differentiating CW tracheids. AGX labeling began to be detected in the corner of the S(1) layer at the early S(1) formation stage. Subsequently, the cell corner middle lamella (ccML) showed strong AGX labeling when intercellular spaces were not fully formed. AGX labeling was uniformly distributed in the S(1) layer, but showed uneven distribution in the S(2) layer. AGX labeling was mainly detected in the inner S(2) layer after the beginning of the helical cavity formation. The outer S(2) layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S(2) layer. These patterns of AGX labeling in the S(2) layer opposed the lignin and β-1-4-galactan distribution in CW tracheids. GGM labeling patterns were almost identical to AGX labeling in the early stages of CW tracheids, and GGM labeling was detected in the entire S(2) layer from the early S(2) formation stage of CW tracheids with some spatial differences in labeling density depending on developmental stage. Compared with NW tracheids, CW tracheids showed significantly different AGX distributions in the secondary cell wall but similar GGM labeling patterns. No significant differences were observed in labeling after delignification of CW tracheids.

We report a simple analytical procedure combining near-infrared (NIR) spectroscopy with multivariate analysis to detect the saccharification efficiency of pretreated rice straw. Three types of sample preparation methods were tested to develop a powerful calibration model, with the disk sample used as the standard protocol. From the spectra dataset of NaOH-treated biomass, we obtained a good calibration for the saccharification ratio and some major structural components by partial least-squares regression. Adding dataset from hot water and dilute sulfuric acid pretreatments to NaOH sample dataset, an acceptable calibration model to predict the saccharification ratio as well as the glucose, xylose, and lignin contents was generated. NIR has a great potential for rapid screening of saccharification efficiency of pretreated biomass, which would allows us to control the quality of processing toward better bioethanol production.

Wood is composed of various types of cells and each type of cell has different structural and functional properties. However, the temporal and spatial diversities of cell wall components in the cell wall between different cell types are rarely understood. To extend our understanding of distributional diversities of cell wall components among cells, we investigated the immunolabeling of mannans (O-acetyl-galactoglucomannans, GGMs) and xylans (arabino-4-O-methylglucuronoxylans, AGXs) in ray cells and pits. The labeling of GGMs and AGXs was temporally different in ray cells. GGM labeling began to be detected in ray cells at early stages of S(1) formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S(2) formation stage in tracheids. The occurrence of GGM and AGX labeling in ray cells was also temporally different from that of tracheids. AGX labeling began to be detected much later in ray cells than in tracheids. GGM labeling also began to be detected in ray cells either slightly earlier or later than in tracheids. In pits, GGM labeling was detected in bordered and cross-field pit membranes at early stages of pit formation, but not observed in mature pits, indicating that enzymes capable of GGM degradation may be involved in pit membrane formation. In contrast to GGMs, AGXs were not detected in pit membranes during the entire developmental process of bordered and cross-field pits. AGXs showed structural and depositional variations in pit borders depending on the developmental stage of bordered and cross-field pits.

In this study, saccharification of the inner bark of Eucalyptus was carried out by enzymatic hydrolysis to produce bioethanol from non-food biomass. To enhance the accessibility of the enzyme to the polysaccharides such as cellulose and holocellulose in the cell wall of the bark, the bark was subjected to hydrothermal pre-treatment with carbon dioxide. This pre-treatment considerably influenced enzymatic hydrolysis. The main component (over 90%) of the generated monosaccharide was glucose, and the yield of glucose on the basis of alpha-cellulose reaches about 80%. This result suggests that the secondary wall, whose main component is cellulose, was effectively hydrolyzed by the enzyme. Microscopic analysis revealed that after pre-treatment, the phloem parenchyma cell had a considerably swollen primary wall and the phloem fibre showed many nano-clefts within its secondary wall. These structural changes appeared to promote enzymatic hydrolysis, because of high accessibility of enzymes to cellulose in the secondary wall.

We investigated the deposition of glucomannans (GMs) in differentiating earlywood tracheids of Cryptomeria japonica using immunocytochemical methods. GMs began to deposit at the corner of the cell wall at the early stages of S(1) formation and showed uneven distribution in the cell wall during S(1) formation. At the early stages of S(2) formation, limited GM labeling was observed in the S(2) layer, and then the labeling increased gradually. In mature tracheids, the boundary between the S(1) and S(2) layers and the innermost part of the cell wall showed stronger labeling than other parts of the cell wall. Deacetylation of GMs with mild alkali treatment led to a significant increase in GM labeling and a more uniform distribution of GMs in the cell wall than that observed before deacetylation, indicating that some GM epitopes may be masked by acetylation. However, the changes in GM labeling after deacetylation were not very pronounced until early stages of S(2) formation, indicating that GMs deposited in the cell wall at early stages of cell-wall formation may contain fewer acetyl groups than those deposited at later stages. Additionally, the density of GM labeling increased in the cell wall in both specimens before and after GM deacetylation, even after cell-wall formation was complete. This finding suggests that some acetyl groups may be removed from GMs after cell-wall formation is complete as part one of the tracheid cell aging processes.

Compression wood (CW) contains higher quantities of beta-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of beta-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of beta-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of beta-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S(1) layer. At this stage, CW was strongly labeled in the S(1) layer, whereas no label was observed in the S(1) layer of NW and OW. Immunogold labeling showed that beta-1-4-galactan in the S(1) layer of CW tracheids significantly decreased during the formation of the S(2) layer. Most beta-1-4-galactan labeling was present in the outer S(2) region in mature CW tracheids, and was absent in the inner S(2) layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of beta-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask beta-1-4-galactan epitopes. The study clearly showed that beta-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.