広葉樹ＣＴＭＰの製造 ＩＩ バイサルファイト前処理ＣＭＴＰのリファイニングによる形態的変化

The fine structure and water permeability of potato tuber periderm have been studied. Periderm membranes (PM) were isolated enzymatically using pectinase and cellulase. They were composed of, about six layers of phellem cells arranged in radial rows. The walls of phellem cells consist of cellulosic primary and tertiary walls and suberized secondary walls which are lamellated. Middle lamellae and primary walls contain lignin. Since the PM did not disintegrate during enzymatic isolation it appears that lignin also extends into the secondary suberized walls. The water permeability of PM was low, ranging from 1-3·10(-10) m s(-1). This low water permeability developed only during storage of tubers in air. Periderm membranes from freshly harvested tubers had a relatively high permeability. The low permeability of PM from stored tubers is attributed to soluble lipids associated with suberin since: (1) extraction of soluble lipids from PM increased permeability by more than 100-fold, (2) a phase transition of soluble lipids was observed between 46 and 51° C, and (3) only the permeability of PM decreased during storage while the permeability of extracted PM remained unchanged. Evidence is presented that two pathways for water movement exist in parallel. Pathway 1 is represented by middle lamellae and primary walls extending in radial direction across the membranes. This pathway has a relatively high specific permeability. Pathway 2 is represented by a polylaminated structure made up of tangential walls of phellem cells which are orientated normal to the direction of water flow. This pathway has a low specific permeability because of the properties of secondary walls incrusted with soluble lipids. It is calculated that about 10% of the water flows across pathway 1 and 90% across pathway 2 which has a volume fraction of 0.995.

Primary cell walls are deposited and remodeled during cell division and expansion. Secondary cell walls are deposited in specialized cells after the expansion phase. It is presently unknown whether and how these processes are interrelated. The Arabidopsis (Arabidopsis thaliana) MUR10 gene is required for normal primary cell wall carbohydrate composition in mature leaves as well as for normal plant growth, hypocotyl strength, and fertility. The overall sugar composition of young mur10 seedlings is not significantly altered; however, the relative proportion of pectin side chains is shifted toward an increase in 1 --> 5-alpha-arabinan relative to 1 --> 4-beta-galactan. mur10 seedlings display reduced fucogalactosylation of tightly cell wall-bound xyloglucan. Expression levels of genes encoding either nucleotide sugar interconversion enzymes or glycosyl transferases, known to be involved in primary and secondary cell wall biosynthesis, are generally unaffected; however, the CesA7 transcript is specifically suppressed in the mur10-1 allele. The MUR10 locus is identical with the CesA7 gene, which encodes a cellulose catalytic subunit previously thought to be specifically involved in secondary cell wall formation. The xylem vessels in young mur10 hypocotyls are collapsed and their birefringence is lost. Moreover, a fucogalactosylated xyloglucan epitope is reduced and a 1 --> 5-alpha-arabinan epitope increased in every cell type in mur10 hypocotyls, including cells that do not deposit secondary walls. mur10 also displays altered distribution of an arabinogalactan-protein epitope previously associated with xylem differentiation and secondary wall thickening. This work indicates the existence of a mechanism that senses secondary cell wall integrity and controls biosynthesis or structural remodeling of primary cell walls and cellular differentiation.

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

Proline-rich protein gene PdPRP regulates secondary wall formation in poplar

Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production.

Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy.

Plant cells are surrounded by a carbohydrate-rich extracellular matrix known as the cell wall. Primary cell walls are laid down around dividing and elongating cells and consist largely of the polysaccharides cellulose, hemicelluloses, and pectin along with approximately 10% protein. Specific cells such as xylem vessels and fibers lay down a secondary wall rich in cellulose, hemicellulose, and lignin, with lesser amounts of pectin. Most of the models depict the plant cell wall as a matrix of separate polysaccharides. However, the recent identification of a proteoglycan that contains covalently attached pectin and xylan indicates that at least some of these wall glycans exist as domains within a single glycopolymer and that current models of the wall need to be revised. Furthermore, several cell wall biosynthesis mutants, including the secondary cell wall mutant irregular xylem (irx) 8, are affected in multiple cell wall polymers making it challenging to define the biochemical function of the mutated gene. The goal of this review is to provide a background for studying genes which encode secondary cell wall biosynthetic proteins whose mutation affects multiple wall polymers including xylan and lignin. We first review the phenotypes of the irx mutants and then summarize the current understanding of the structure and synthesis of xylan and lignin along with a review of transcription factors known to affect secondary wall synthesis. This review is intended to serve as a resource for those studying genes that encode proteins involved in the synthesis of plant secondary wall lignin and xylan.

Early responses of legume roots toRhizobium inoculation include new cell wall synthesis and induction of some putative wall protein genes. Although the predicted amino acid sequences of several early nodulins indicate that they encode proline-rich proteins (PRPs), the proteins have been neither isolated nor has their presence been demonstrated in cell walls. We have used polyclonal antibodies against PRP2 from soybean to identify and localize proline-rich proteins in pea nodules. On immunoblots, several PRPs were detected, ranging from less than 20 kDa to 110 kDa. Immunocytochemistry revealed that tissues of the vascular cylinder contained abundant PRPs, particularly in the secondary cell walls of xylem elements and phloem fibers. PRPs were also found within the primary wall of the nodule endodermis and within Casparian strips of the vascular endodermis. Of symbiotic importance, PRPs were a prominent component of the infection thread matrix in newly infected root cells and in nodules. PRPs were also secreted by cells in the uninfected nodule parenchyma, where they were found occluding intercellular spaces outside the middle lamella. Despite structural conservation among members of this class of cell wall proteins, PRPs were targeted to distinct layers of the extracellular matrix dependent upon cell type, and may thus play separate roles in the biology of plant cells. The putative functions and the potential for interactions between PRPs and other wall polymers are discussed.

The lignin distribution between the middle lamella and the cell wall of spruce fibers has been determined by a new technique based on a mercurization of the lignin and a concomitant determination of mercury by the SEM-EDXA technique. The ratio of lignin in the middle lamella at the cell corners to the lignin in the secondary wall was 2.5±0.6 for latewood and 2.4±0.6 for earlywood. This gives a lignin content of 55–58% in the true middle lamella in the cell corners. The reactivity to mercuric acetate of different wood elements was determined in separate experiments. Fractions enriched in ray cells, middle lamella, and compression wood all reacted at the same rate as the whole wood; about one mole of mercury was incorporated per mole of lignin (C9-unit).

The formation of cavities in the secondary wall (S 2 ) of fibres and tracheids was revealed in wood artificially incubated and naturally infected by a range of wood degrading basidiomycetes. Cavity formation is a characteristic feature of soft rot attack. the observed phenomena however differed from a soft rot mode of attack as hyphal growth was not ascertained within the cell walls. Atypical for a soft rot. or any commonly described white rot degradation pattern, extensive delignification always preceded cavity formation. Single cavities were separated from another within the secondary wall (S 2 ) by peculiar conspicuous radial structures, which apparently have not been described in white-rotted wood before. Radial structures were orientated perpendicular to the middle lamella and extended from the S 1 to the S 3 . Like the S 3 , the exposed radial structures, which revealed birefringence when viewed between crossed nicols, showed an inherent resistance to degradation by the selected basidiomycetes. The presence of cellulose within radial structures of the delignified secondary walls (S 2 ) was also confirmed by staining techniques.

The effect of xylanase treatment of eucalyptus wood chips on chip refining and fiber properties was investigated. The fiber separation region and fiber surface structure were observed with SEM, TEM, and AFM. The fiber length and fines were analyzed with a Bauer-McNett classifier and optical image analysis of flowing suspensions (FQA). The results showed that xylanase degraded and hydrolyzed some xylan in the fiber wall, thus loosening the fiber wall structure. Therefore, in the subsequent refining process, fiber separation occurred in the secondary wall. This resulted in fibers with less lignin and extractives on the surface, which will benefit the interfiber bonding.

Xyloglucan is the major hemicellulose of dicotyledon primary cell walls, affecting the load-bearing framework with the participation of xyloglucan endo-transglycosylase/hydrolases (XTHs). We used loss- and gain-of function approaches to study functions of XTH4 and XTH9 abundantly expressed in cambial regions during secondary growth of Arabidopsis (Arabidopsis thaliana). In secondarily thickened hypocotyls, these enzymes had positive effects on vessel element expansion and fiber intrusive growth. They also stimulated secondary wall thickening but reduced secondary xylem production. Cell wall analyses of inflorescence stems revealed changes in lignin, cellulose, and matrix sugar composition indicating an overall increase in secondary versus primary walls in mutants, indicative of higher xylem production compared with the wild type (since secondary walls were thinner). Intriguingly, the number of secondary cell wall layers compared with the wild type was increased in xth9 and reduced in xth4, whereas the double mutant xth4x9 displayed an intermediate number of layers. These changes correlated with specific Raman signals from the walls, indicating changes in lignin and cellulose. Secondary walls were affected also in the interfascicular fibers, where neither XTH4 nor XTH9 was expressed, indicating that these effects were indirect. Transcripts involved in secondary wall biosynthesis and cell wall integrity sensing, including THESEUS1 and WALL ASSOCIATED KINASE2, were highly induced in the mutants, indicating that deficiency in XTH4 and XTH9 triggers cell wall integrity signaling, which, we propose, stimulates xylem cell production and modulates secondary wall thickening. Prominent effects of XTH4 and XTH9 on secondary xylem support the hypothesis that altered xyloglucan affects wood properties both directly and via cell wall integrity sensing.

Three cottons differing in their extent of fuzz fibers (linters) and final length of lint fibers were analyzed for amount of fiber cell walls and fiber cellulose at various times postanthesis. Cellulose determinations were performed directly on whole fibers and on fiber cell wall preparations. The data suggest that the presence of fuzz fibers does not account for a rise, followed by a plateau, followed by a rise, in cellulose content expressed as a percentage of cell wall material. It is concluded that: (1) under our greenhouse conditions, all fuzz fibers are initiated by day eight after anthesis; (2) weight per mm length of all fibers increases up to the point of secondary wall deposition and increases even more rapidly after that; (3) deposition of secondary wall cellulose in fuzz fibers probably does not begin until after similar deposition begins in lint fibers; (4) the actual amount of cellulose in primary walls of all elongating fibers (fuzz and lint) is a constant value, about 1 × 10−16 mg/mm; and (5) secondary wall cellulose deposition in lint fibers begins very sharply, in advance of cessation of elongation, at a time closely related to final lint fiber length. It is speculated that: (1) cell wall preparation procedures may remove significant amounts of noncellulosic wall material, thus making it difficult to define all functional constituents on the basis of what is left in a cell wall residue; and/or (2) primary walls may lose to the cytoplasm some of their constituents in advance of secondary wall deposition, the extent of loss varying due to developmental age of the elongating fibers.

The composition of the cell wall of the cotton fiber (Gossypium hirsutum L. Acala SJ-1) has been studied from the early stages of elongation (5 days postanthesis) through the period of secondary wall formation, using cell walls derived both from fibers developing on the plant and from fibers obtained from excised, cultured ovules. The cell wall of the elongating cotton fiber was shown to be a dynamic structure. Expressed as a weight per cent of the total cell wall, cellulose, neutral sugars (rhamnose, fucose, arabinose, mannose, galactose, and noncellulosic glucose), uronic acids, and total protein undergo marked changes in content during the elongation period. As a way of analyzing absolute changes in the walls with time, data have also been expressed as grams component per millimeter of fiber length. Expressed in this way for plant-grown fibers, the data show that the thickness of the cell wall is relatively constant until about 12 days postanthesis; after this time it markedly increases until secondary wall cellulose deposition is completed. Between 12 and 16 days postanthesis increases in all components contribute to total wall increase per millimeter fiber length. The deposition of secondary wall cellulose begins at about 16 days postanthesis (at least 5 days prior to the cessation of elongation) and continues until about 32 days postanthesis. At the time of the onset of secondary wall cellulose deposition, a sharp decline in protein and uronic acid content occurs. The content of some of the individual neutral sugars changes during development, the most prominent change being a large increase in noncellulosic glucose which occurs just prior to the onset of secondary wall cellulose deposition. Methylation analyses indicate that this glucose, at least in part, is 3-linked. In contrast to the neutral sugars, no significant changes in cell wall amino acid composition are observed during fiber development.Compositional analyses of cell walls derived from culture-grown fibers indicate that these walls are remarkably similar to those derived from fibers grown on the plant, both in terms of composition and in terms of relative changes in composition during development.A comparison of our results on total cell wall composition and linkages of sugars as determined by a preliminary methylation analysis of unfractionated fiber walls indicates that the primary cell wall of cotton fibers is similar to that of primary cell walls of other dicotyledons and of gymnosperms as reported in the literature.

Cotton fiber is an important natural textile fiber due to its exceptional length and thickness. These properties arise largely through primary and secondary cell wall synthesis. The cotton fiber of commerce is a cellulosic secondary wall surrounded by a thin cuticulated primary wall, but there were only sparse details available about the polysaccharides in the fiber cell wall of any cotton species. In addition, Gossypium hirsutum (Gh) fiber was known to have an adhesive cotton fiber middle lamella (CFML) that joins adjacent fibers into tissue-like bundles, but it was unknown whether a CFML existed in other commercially important cotton fibers. We compared the cell wall chemistry over the time course of fiber development in Gh and Gossypium barbadense (Gb), the two most important commercial cotton species, when plants were grown in parallel in a highly controlled greenhouse. Under these growing conditions, the rate of early fiber elongation and the time of onset of secondary wall deposition were similar in fibers of the two species, but as expected the Gb fiber had a prolonged elongation period and developed higher quality compared to Gh fiber. The Gb fibers had a CFML, but it was not directly required for fiber elongation because Gb fiber continued to elongate rapidly after CFML hydrolysis. For both species, fiber at seven ages was extracted with four increasingly strong solvents, followed by analysis of cell wall matrix polysaccharide epitopes using antibody-based Glycome Profiling. Together with immunohistochemistry of fiber cross-sections, the data show that the CFML of Gb fiber contained lower levels of xyloglucan compared to Gh fiber. Xyloglucan endo-hydrolase activity was also higher in Gb fiber. In general, the data provide a rich picture of the similarities and differences in the cell wall structure of the two most important commercial cotton species.