Quantitative morphological transformation of vascular bundles in the culm of moso bamboo (Phyllostachys pubescens).

Vascular bundles of bamboo are determinants for mechanical properties of bamboo material and for physiological properties of living bamboo. The morphology of vascular bundles reflecting mechanical and physiological functions differs not only within internode tissue but also among different internodes in the culm. Although the distribution of vascular bundle fibers has received much attention, quantitative evaluation of the morphological transformation of vascular bundles associated with spatial distribution patterns has been limited. In this study deep learning models were used to determine quantitative changes in the distribution and morphology of vascular bundles in the culms of moso bamboo (Phyllostachys pubescens). A precise model for extracting vascular bundles from cross-sectional images was constructed using the U-Net model. Analyses of extracted vascular bundles from different internodes showed significant changes in vascular bundle distribution and morphology among internodes. Vascular bundles in lower internodes showed outer relative position and larger area than those in upper internodes. Aspect ratio and eccentricity indicate that vascular bundles in internodes near the base have more elliptical morphology, with a long axis in the radial direction. The variational autoencoder model using extracted vascular bundles enabled simulation of the morphological transformation of vascular bundles along with radial direction. These deep learning models enabled highly accurate quantification of vascular bundle morphologies, and will contribute to a further understanding of bamboo development as well as evaluation of the mechanical and physiological properties of bamboo.

Formation of xylem tissues and secondary cell walls is diminished by severe and consecutive insect defoliation.

PREMISE: Insect defoliation of trees causes unusual changes to wood anatomy and slows radial growth that decreases tree value; however, the characteristics of these anatomical changes in hardwoods remain unclear. The aim of this study was to characterize the anatomy and histochemistry of the wood in trunks of Betula maximowicziana trees after severe insect defoliation. METHODS: Secondary xylem tissues were sampled from trunks that had been defoliated by Caligula japonica at Naie and Furano in central Hokkaido during 2006-2012, then cross-dated and examined microscopically and stained histochemically to characterize anatomical and chemical changes in the cells. RESULTS: White rings with thin-walled wood fibers and greatly reduced annual ring width in the subsequent year were observed in samples from both sites. From these results, the year that the white rings formed was determined, and severe defoliation was confirmed to trigger white ring formation. The characteristics may prove useful to detect the formation year of white rings. Scanning electron microscopy and histochemical analyses of the white rings indicated that the thickness of the S2 layer in the wall of wood fiber cells decreased, but xylan and lignin were still deposited in the cell walls of wood fibers. However, the walls of the fibers rethickened after the defoliation. CONCLUSIONS: Our results suggest that B. maximowicziana responds to a temporary lack of carbon inputs due to insect defoliation by regulating the thickness of the S2 layer of the cell wall of wood fibers. For B. maximowicziana, insect defoliation late in the growing season has serious deleterious effects on wood formation and radial growth.

Culture Conditions for Mycelial Growth and Anti-Cancer Properties of Termitomyces.

Termitomyces sp. that grow in symbiosis with fungus-farming Termites have medicinal properties. However, they are rare in nature, and their artificial culture is challenging. The expression of AXL receptor tyrosine kinase and immune checkpoint molecules favor the growth of cancer cells. The study evaluated the optimal conditions for the artificial culture of Termitomyces and their inhibitory activity on AXL and immune checkpoint molecules in lung adenocarcinoma and melanoma cell lines. The culture of 45 strains of Termitomyces was compared. Five strains with marked growth rates were selected. Four of the selected strains form a single cluster by sequence analysis. The mycelium of 4 selected strains produces more fungal mass in potato dextrose broth than in a mixed media. The bark was the most appropriate solid substrate for Termitomyces mycelia culture. The mycelium of all five selected strains showed a higher growth rate under normal CO2 conditions. The culture broth, methanol, and ethyl acetate of one selected strain (T-120) inhibited the mRNA relative expression of AXL receptor tyrosine kinase and immune checkpoint molecules in cancer cell lines. Overall, these results suggest the potential usefulness of Termitomyces extracts as a co-adjuvant therapy in malignant diseases.

Upregulation of MAP kinase HOG1 gene of white-rot fungus Phlebia sp. MG-60 inhibits the ethanol fermentation and mycelial growth.

Wood biomass conversion for fossil resource replacement could result in the sustainable production of chemicals, although lignin represents an obstacle to efficient polysaccharide use. White-rot fungus Phlebia sp. MG-60 reportedly selectively and aerobically degrades lignin in hardwood, then it begins cellulose saccharification from the delignified wood to produce ethanol. Environmental conditions might change white-rot fungi-driven biomass conversion. However, how the environmental response sensor affects ethanol fermentation in white-rot fungi remains elusive. In this study, we focused on MGHOG1, the yeast Hog1 homolog in Phlebia sp. MG-60, a presumably important player in osmoresponse. We generated MGHOG1 overexpressing (OE) transformants in Phlebia sp. MG-60, exhibiting slower mycelial growth compared with the wild-type under salinity stress. MGHOG1 overexpressing liquid cultures displayed suppressed mycelial growth and ethanol fermentation. Therefore, MGHOG1 potentially influences ethanol fermentation and mycelial growth in Phlebia sp. MG-60. This study provides novel insights into the regulation of white-rot fungi-mediated biomass conversion.

Deposition patterns of feruloylarabinoxylan during cell wall formation in moso bamboo.

MAIN CONCLUSION: The feruloylarabinoxylan deposition was initiated at the formation of the secondary cell wall, especially S2 layer in moso bamboo, which may affect crosslinking between cell wall components and plant growth. Hemicelluloses, major components of plant cell walls that are hydrogen bonded to cellulose and covalently bound to lignin, are crucial determinants of cell wall properties. Especially in commelinid monocotyledons, arabinoxylan is often esterified with ferulic acid, which is essential to crosslinking with cell wall components. However, the deposition patterns and localization of ferulic acid during cell wall formation remain unclear. In this study, developing moso bamboo (Phyllostachys pubescens) culms were used to elucidate deposition patterns of hemicelluloses including feruloylarabinoxylan. Ferulic acid content peaked with cessation of elongation growth, and thereafter decreased and remained stable as culm development proceeded. During primary cell wall (PCW) formation, xyloglucan and (1,3;1,4)-ß-glucan signals were detected in all tissues. Along with culm development, arabinoxylan and feruloylarabinoxylan signals were sequentially observed in the protoxylem, vascular fibers and metaxylem, and parenchyma. Feruloylarabinoxylan signals were observed slightly later than arabinoxylan signals. Arabinoxylan signals were observed throughout the compound middle lamella and secondary cell wall (SCW), whereas the feruloylarabinoxylan signal was localized to the S2 layer of the SCW. These results indicate that the biosynthesis of hemicelluloses is regulated in accordance with cell wall layers. Feruloylarabinoxylan deposition may be initiated at the formation of SCW, especially S2 layer formation. Ferulic acid-mediated linkages of arabinoxylan-arabinoxylan and arabinoxylan-lignin would arise during SCW formation with the cessation of elongation growth.

Active Transport of Lignin Precursors into Membrane Vesicles from Lignifying Tissues of Bamboo.

Lignin is the second most abundant natural polymer on Earth and is a major cell wall component in vascular plants. Lignin biosynthesis has three stages: biosynthesis, transport, and polymerization of its precursors. However, there is limited knowledge on lignin precursor transport, especially in monocots. In the present study, we aimed to elucidate the transport mode of lignin monomers in the lignifying tissues of bamboo (Phyllostachys pubescens). The growth manners and lignification processes of bamboo shoots were elucidated, which enabled us to obtain the lignifying tissues reproducibly. Microsomal membrane fractions were prepared from tissues undergoing vigorous lignification to analyze the transport activities of lignin precursors in order to show the ATP-dependent transport of coniferin and p-glucocoumaryl alcohol. The transport activities for both precursors depend on vacuolar type H+-ATPase and a H+ gradient across the membrane, suggesting that the electrochemical potential is the driving force of the transport of both substrates. These findings are similar to the transport properties of these lignin precursors in the differentiating xylem of poplar and Japanese cypress. Our findings suggest that transport of coniferin and p-glucocoumaryl alcohol is mediated by secondary active transporters energized partly by the vacuolar type H+-ATPase, which is common in lignifying tissues. The loading of these lignin precursors into endomembrane compartments may contribute to lignification in vascular plants.

Prior secondary cell wall formation is required for gelatinous layer deposition and posture control in gravi-stimulated aspen.

Cell walls, especially secondary cell walls (SCWs), maintain cell shape and reinforce wood, but their structure and shape can be altered in response to gravity. In hardwood trees, tension wood is formed along the upper side of a bending stem and contains wood fiber cells that have a gelatinous layer (G-layer) inside the SCW. In a previous study, we generated nst/snd quadruple-knockout aspens (Populus tremula × Populus tremuloides), in which SCW formation was impaired in 99% of the wood fiber cells. In the present study, we produced nst/snd triple-knockout aspens, in which a large number of wood fibers had thinner SCWs than the wild type (WT) and some had no SCW. Because SCW layers are always formed prior to G-layer deposition, the nst/snd mutants raise interesting questions of whether the mutants can form G-layers without SCW and whether they can control their postures in response to changes in gravitational direction. The nst/snd mutants and the WT plants showed growth eccentricity and vessel frequency reduction when grown on an incline, but the triple mutants recovered their upright growth only slightly, and the quadruple mutants were unable to maintain their postures. The mutants clearly showed that the G-layers were formed in SCW-containing wood fibers but not in those lacking the SCW. Our results indicate that SCWs are essential for G-layer formation and posture control. Furthermore, each wood fiber cell may be able to recognize its cell wall developmental stage to initiate the formation of the G-layer as a response to gravistimulation.

Proton Gradient-Dependent Transport of p-Glucocoumaryl Alcohol in Differentiating Xylem of Woody Plants.

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.

Down-regulation of pyruvate decarboxylase gene of white-rot fungus Phlebia sp. MG-60 modify the metabolism of sugars and productivity of extracellular peroxidase activity.

Ethanologenic white-rot fungus Phlebia sp. MG-60-P2 produces ethanol directly from several lignocelluloses. Efficient gene silencing methods are needed for metabolic engineering of this fungus for biorefinery use. In this study, we evaluated the effectiveness of RNAi-mediated silencing of the pyruvate decarboxylase gene of Phlebia sp. MG-60-P2 (MGpdc1). The RNAi lines generated showed a variety of suppression levels of ethanol production and MGpdc1 expression, and two selected strains led to different metabolic fluxes, resulting in rapid accumulation of xylitol from xylose. Knockdown lines KD2 and KD10 showed different strength of silencing. The moderate-inhibition line (KD10) showed faster xylitol accumulation from xylose than the severe-inhibition line (KD2). KD2, KD10 and knockout line KO77 showed higher extracellular peroxidase activity than the wild-type. Gene silencing using RNAi for MGpdc1 in the ethanologenic white-rot fungus Phlebia sp. MG-60-P2 is an effective first step in metabolic engineering to produce other chemicals besides ethanol. This high efficiency of transformation and silencing effect will make it possible to cotransform with multiple expression vectors which enhance the minor metabolic pathway or introduce exogenous metabolic reaction in Phlebia sp. MG-60-P2.

Accumulation of sugar from pulp and xylitol from xylose by pyruvate decarboxylase-negative white-rot fungus Phlebia sp. MG-60.

Phlebia sp. MG-60 is a white-rot fungus that produces ethanol with high efficiency from lignocellulosic biomass without additional enzymes. Through engineering of this powerful metabolic pathway for fermentation in Phlebia sp. MG-60, chemical compounds other than ethanol could be produced. Here, we demonstrate sugar accumulation from unbleached hardwood kraft pulp and conversion of xylose to xylitol by pyruvate decarboxylase (pdc)-negative Phlebia sp. MG-60. We isolated Phlebia sp. strain MG-60-P2 from protoplasts to unify the protoplast phenotypes of the regenerated strains. Homologous recombination achieved a stable pdc-knockout line, designated KO77. The KO77 line produced traces of ethanol, but accumulated xylitol from xylose or glucose from unbleached hardwood kraft pulp. These metabolic changes in the pdc-knockout strain reflect the potential of metabolic engineering in Phlebia sp. MG-60 for direct production of chemical compounds from lignocellulosic biomass.

Proton-dependent coniferin transport, a common major transport event in differentiating xylem tissue of woody plants.

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.