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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.

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.

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.

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

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.

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.

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.

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.

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.

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 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.

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.

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.

The cellular distribution of heartwood substances and the structure of the pathways for their diffusion were studied in Acacia mangium Willd. Apart from ray parenchyma cells, axial parenchyma cells also are involved in the formation of heartwood substances. Heartwood substances were unevenly distributed in the heartwood. A closer inspection of interfibre pit pairs revealed that, although many pit membranes were completely covered with encrusting materials, some pit pairs had many small openings on their pit membranes. The openings possibly function as intercellular diffusion pathways for heartwood substances. The sizes of the pits varied considerably, ranging from 0.4 to 2.3 μm in diameter. These structural variations in the interfiber pits might be one of the factors contributing to the uneven distribution of the heartwood substances. A large number of blind pits were present in the ray parenchyma cells and faced the intercellular spaces, into which heartwood substances from the ray parenchyma cells were released via these blind pits. Resin-cast replicas demonstrated that the intercellular spaces and the blind pits formed a three-dimensional network that is considered to serve as an extracellular diffusion pathway for heartwood substances.

We investigated radial resin canals in Pinus densiflora by means of serial tangential sections taken from phloem to xylem through the cambium. The canals were found within fusiform rays in both phloem and xylem. The ducts were closed in the cambial zone, but opened at widely differing distances from the cambium among individual radial resin canals, ranging from 120 to 340 μm on the phloem side and from 260 to 640 μm on the xylem side. Further, the ducts were not open continuously on both sides. The average number of radial resin canals in the tangential plane was 0.76/mm2. In the cambial zone, central cells of fusiform rays which might develop into epithelial cells later, were smaller and more deeply stained than the surrounding ray initial cells, allowing them to be identified at the initial stage. Two or more radial resin canals situated nearby each other were connected through an axial resin canal.

The distributions of syringyl lignin and glucuronoxylans in contact and non-contact wood fibers of Pterocymbium beccarii, Paulownia tomentosa, and Albizia julibrissin were investigated using the Mäule color reaction and immunogold labeling, respectively. In the Mäule color reaction, the fibers with small diameters (tip regions of wood fibers) appeared much darker in color than those with larger diameters (body regions of wood fibers), but there were no significant differences in the depth of color between the contact and non-contact wood fibers with similar diameters. This finding suggests that the tips of the wood fibers tend to be richer in syringyl lignin than the bodies, but there is no positive correlation between the content of syringyl lignin and the distance of the wood fiber from the ray. In the immunogold labeling, no essential difference was found in the labeling distribution and density between contact and non-contact wood fibers in all three species. In Albizia julibrissin, moreover, almost all wood fibers regardless of contact or non-contact with the rays, contained starch grains in the outer portion of the outermost annual ring. These findings indicate that the distance of wood fibers from the rays do not affect the transport of photosynthate, so that even the wood fibers far away from the rays are supplied with almost the same amount of photosynthetic products, which are a source of raw materials for cell wall biosynthesis.

A three-dimensional network of intercellular spaces, deemed an extracellular diffusion pathway for heartwood substances, was detected in Albizia julibrissin. Electron microscopy revealed a large number of blind pits in ray parenchyma cells, most facing intercellular spaces. Heartwood substances, which are synthesized in the ray parenchyma cells, are released not only into neighboring cells through pit pairs, but also into the intercellular spaces through the blind pits. There were two types of wood fiber in the heartwood region: one with a lumen surface lined with heartwood substances and one that lacked such lining on its lumen surface. This finding supports the observation that the cellular distribution of heartwood substances is not homogenous throughout the heartwood. There was no positive correlation, however, between this uneven distribution and the distance of a wood fiber from the ray.

Proportions ofaxial elements without any spatial contacts with rays were investigated. Non-contact rates between rays and fusiforrn initials or axial parenchyma strands were examined in tangential sections of cambial or axial parenchyma zones in 21 hardwood species . The noncontact rates were very different among species, ranging from 0% to 76%. Serial trans verse sections of xylem were used to deterrnine the non-contact rates between rays and wood fibers in 3 of the 21 hardwood species having high non-contact rates of parenchyma strands . The noncontact rate of wood fibers was 49% in Albizia julibrissin (non-storied cambium), 30% in Paulownia tomentosa (non-storied cambium), and 52% in Pterocymbium beccarii (storied cambium). Tangential seetions of three softwood species, Pinus thunbergii, Cryptomeria japonica, and Chamaecyparis obtusa were used to investigate the contacts between rays and tracheids. All tracheids in the three species had contact with at least one ray, ranging from 1 to 15 rays.

We have identified and characterized novel types of ferredoxin and ferredoxin reductase from Arabidopsis. Among a number of potential ferredoxin reductase genes in the Arabidopsis genome, AtMFDR was identified to encode a homologue of mitochondrial ferredoxin reductase, and AtMFDX1 and AtMFDX2 were predicted to code for proteins similar to mitochondrial ferredoxin. First, we isolated cDNAs for these proteins and expressed them in heterologous systems of insect cells and Escherichia coli, respectively. The recombinant AtMFDX1 and AtMFDR proteins exhibited spectral properties characteristic of ferredoxin and ferredoxin reductase, respectively, and a pair of recombinant AtMFDX1 and AtMFDR proteins was sufficient to transfer electrons from NAD(P)H to cytochrome c in vitro. Subcellular fractionation analyses suggested membrane association of AtMFDR protein, and protein-gel blot analyses and transient expression studies of green fluorescence protein fusions indicated mitochondrial localization of AtMFDX1 and AtMFDR. RNA-gel blot analyses revealed that the accumulation levels of AtMFDXs and AtMFDR gene transcripts were specifically high in flowers, while protein-gel blot analysis demonstrated substantial accumulation of AtMFDR protein in leaf, stem, and flower. Possible physiological roles of these mitochondrial electron transfer components are discussed in relation to redox dependent metabolic pathways in plants.

Lignin distribution in the tracheid cell wall of mild compression wood in Pinus radiata D. Don was examined by interference microscopy, confocal fluorescence microscopy, and ultraviolet (UV) microscopy. Two anatomically different samples of mild compression wood were compared with a sample of normal wood using quantitative interference microscopy and microdensitometry combined with confocal fluorescence microscopy to estimate the quantitative or semiquantitative lignin distribution in the S2 and S2L regions of the secondary cell wall and of the cell corner middle lamella (CCML). One of these samples was briefly examined by UV microscopy for comparison. Quantitative interference microscopy provided information on lignin concentration in different regions of the cell wall with values of 26, 46, and 57%, respectively, for the S2, S2L, and CCML regions of sample 1 and 20, 29, and 46%, respectively, for the same regions of sample 2. Microdensitometry of confocal fluorescence images provided semiquantitative information on the relative lignin distribution based on lignin autofluorescence. Comparison between the two compression wood samples using autofluorescence gave results that were in partial agreement with interference microscopy with respect to the relative lignification levels in the S2, S2L, and CCML regions. Some improvement was achieved by using calibration values for hemicellulose rather than holocellulose for interference data in the S2L region. Results for UV microscopy performed on sample 1 indicated that the lignification of the CCML region was comparable with that of the S2L region in this sample but with some variation among cells. All three techniques indicated significant variation in lignification levels of the S2L and CCML regions among adjacent cells and a significant reduction in the lignification of the CCML region compared to normal wood.Key words: lignin distribution, interference microscopy; confocal fluorescence microscopy, UV microscopy, mild compression wood, Pinus radiata D. Don.

An antiserum against glucuronoxylans (GXs) has been raised from a mouse. The dot-blot immunoassay and competitive inhibition test indicated that the antibodies could bind specifically to GXs. Therefore, the antiserum was used for immunogold labelling to investigate the localization of GXs in Japanese beech. Labelling of GXs was seen only in the secondary walls of xylem cells, but not in the primary walls or the middle lamella. GXs were evenly distributed in the secondary walls except for the outer part of the outer secondary-wall layer in which they were less abundant. The labelling density in each secondary-wall layer (S1, S2, and S3) increased during cell wall formation. This result strongly suggests that the deposition of GXs occurs in a penetrative way.

Laccase has been studied recently in both pure enzymatic, and 'enzyme plus mediator' systems described for the degradation of lignin in wood and pulp. In this study we use TEM immunolabelling probes to label the laccase enzyme and determine where the enzyme is located during wood degradation by Lentinus edodes. Wood degraded by L. edodes was also observed using two-dimensional ultraviolet analysis to determine the extent of lignin degradation, and the relationship between lignin degradation and enzymatic action. Despite extensive degradation of lignin in the wood cell wall and cell corner region of the wood, no penetration of the laccase enzyme into the wood cell wall was observed during the first eight weeks of fungal attack. Degradation of the lignin fraction of the wood cell wall by compounds of lower molecular weight than enzymes is implicated by this work.

Isolated mesophyll cells ofZinnia elegans L. cv. Canary Bird differentiate into tracheary elements in differentiation (D) medium. These elements develop lignified secondary wall thickenings. The influence of 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase (PAL), on lignification ofZinnia tracheary elements was examined. The mesophyll cells were cultured in D and AIP media. The latter medium, in which 100 μM AIP was added to the D medium, inhibited PAL activity, though the differentiation proceeded. Morphological differences of secondary wall thickenings cultured in these two types of media were investigated under an UV microscope and a transmission electron microscope. The secondary wall thickenings at 96 h in the D medium showed strong UV absorption. The fibrillar structure of the thickenings observed clearly at 72 h was covered with electron opaque materials by 96 h. The secondary wall thickenings at 96 h in the AIP medium showed weak UV absorption. The thickenings at 96 h had a cracked appearance. Furthermore, the thickenings showed a little irregular or wavy arrangement of cellulose microfibrils and had many pores and spaces between microfibrils. From these results, the role of lignin accumulation in the formation of secondary wall thickenings was discussed.

Secondary wall thickening is the most characteristic morphological feature of the differentiation of tracheary elements. Isolated mesophyll cells of Zinnia elegans L. cv. Canary Bird in differentiation medium are converted to tracheary elements, which develop lignified secondary wall thickenings. Using this system, we investigated the distribution of two enzymes, phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) and cinnamyl alcohol dehydrogenase (CAD) (EC 1.1.1.195), by both biochemical and immunological methods. Both PAL and CAD appear to be key enzymes in the biosynthesis of lignin precursors, and they have been shown to be associated with the differentiation of tracheary elements. Cultured cells were collected after various times in culture. The culture medium was separated from cells by centrifugation and designated fraction (1), the extracellular fraction. The collected cells were homogenized and separated into four fractions: (2) cytosol; (3) microsomes; (4) cell walls (loosely bound material); and (5) cell walls (tightly bound material). PAL activity was detected in each fraction. The extracellular fraction consistently had the greatest PAL activity. Moreover, PAL activity in the cytosolic fraction increased rapidly prior to lignification, as it did in both the microsomal and the cell wall (tightly bound) fractions during lignification. Antisera against PAL and against CAD detected the proteins with molecular masses that corresponded to those of PAL and CAD in Zinnia. Immuno-electron microscopy revealed that, in differentiating tracheary elements, PAL was dispersed in the cytoplasmic matrix and was located on Golgi-derived vesicles and on the secondary wall thickenings. "Cell-free" immuno-light microscopy supported the putative distribution of PAL on lignifying secondary walls. The pattern of distribution of CAD was similar to that of PAL. Thus, both PAL and CAD seemed to be localized in secondary wall thickenings. From the results of both biochemical assays and immunocytochemical staining, it appeared that at least two types of PAL and CAD are present in differentiating cells. One type of each enzyme is distributed in the cytosol, while the other is secreted from the Golgi apparatus and transported by Golgi-derived vesicles to the secondary wall thickenings.

†Background and Aims Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood. †Methods Eight hardwood species were inclined at 30‐458 at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate. †Key Results Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S1 and S2 layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S1 + S2 + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution. †Conclusions Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.

We investigated the spatial and temporal distribution of xylans in the cell walls of differentiating earlywood tracheids of Cryptomeria japonica using two different types of monoclonal antibodies (LM10 and LM11) combined with immunomicroscopy. Xylans were first deposited in the corner of the S(1) layer in the early stages of S(1) formation in tracheids. Cell corner middle lamella also showed strong xylan labeling from the early stage of cell wall formation. During secondary cell wall formation, the innermost layer and the boundary between the S(1) and S(2) layers (S(1)/S(2) region) showed weaker labeling than other parts of the cell wall. However, mature tracheids had an almost uniform distribution of xylans throughout the entire cell wall. Xylan localization labeled with LM10 antibody was stronger in the outer S(2) layer than in the inner layer, whereas xylans labeled with LM11 antibody were almost uniformly distributed in the S(2) layer. In addition, the LM10 antibody showed almost no xylan labeling in the S(1)/S(2) region, whereas the LM11 antibody revealed strong xylan labeling in the S(1)/S(2) region. These findings suggest that structurally different types of xylans may be deposited in the tracheid cell wall depending on the developmental stage of, or location in, the cell wall. Our study also indicates that deposition of xylans in the early stages of tracheid cell wall formation may be spatially consistent with the early stage of lignin deposition in the tracheid cell wall.

Cellular distribution of coniferin in differentiating xylem of Japanese cypress (Chamaecyparis obtusa) was analyzed by Raman microscopy. Small blocks were collected from differentiating xylem, frozen, cut on their surface with a sliding microtome, and then freeze-dried. Scanning electron microscopy showed numerous needle-like deposits in the tracheid lumina from the beginning of the S1 layer formation to the S2 layer-forming stage. The Raman spectrum of the deposits in the tracheid lumen was similar to that of coniferin. The presence of coniferin in a water extract from differentiating xylem was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy and 1H- and 13C-nuclear magnetic resonance spectra. Differential Raman spectra taken from samples before and after washing with water and dehydration in an ethanol showed that developing secondary walls contained coniferin during the S2 layer-forming stage and also after S3 layer formation. In contrast, coniferin was detected in the cell corner middle lamella during the S2 layer-forming stage, and the differential spectra were different from that of coniferin after S3 layer formation. The differential spectrum in this stage was similar to that of a dehydrogenation polymer of coniferyl alcohol prepared by the “zulauf” method (bulk polymerization). These results suggest that free lignin oligomers of the type bulk polymerizate might exist in the cell corner middle lamella during the S3 layer-forming stage and can be removed from specimens during washing and dehydration. The results can be interpreted in a way that no such oligomer exists in the secondary wall during the same stage owing to endwise addition of monolignols (in analogy to a “zutropf” polymerization).

Microspectrometry is the most definitive technique for obtaining both ultraviolet (UV) and visible light absorption spectra from a very limited area, and this technique allows the determination of lignin distribution throughout an individual cell wall. It is generally accepted that hardwood lignin .is composed mainly of guaiacyl and syringyl moieties. Our microspectrometric investigations revealed variation of lignin distribution within an annual ring in beech (Fagus crenata).

An antisense gene for Aralia cordata cinnamyl alcohol dehydrogenase (CAD) was introduced into tobacco plants. Two transgenic plants showed 55 and 20% reduction of the CAD activity comparedto that of the control plant. Lignin content measured by the acetyl bromide method showed no significant differences between these plants and the control plant. However, the content of p-hydroxycinnamaldehydegroups in lignin was higher in the transgenic than in the control plants. The increase of p-hydroxycinnamaldehydegroups correlated inversely with the levels of CAD activity. Phloroglucinol staining of transverse sections of stem internode indicated that the fiber secondary wall reacted more strongly with the reagentin the transgenic plants. These results suggested that suppression of CAD activity in tobacco plants results in an increase of cinnamaldehyde groups in lignin, but no significant change in lignin content.

Tracheary elements differentiated from isolated Zinnia: mesophyll cells were observed at various times of culture under a scanning electron microscope. Perforation occurred on the primary wall at one of the longitudinal ends in single tracheary elements. In double tracheary elements, which both of two cells derived from a single cell differentiated into, the pore opened on the primary walls both at the junction of the two tracheary elements and at a longitudinal end of one of the two tracheary elements. These results suggest not only that a single tracheary element has its own program to form a perforation at one end without being affected by neighboring cells, but also that isolated cells indeed hold some traces of polarity and cell-cell communication.

Cultured cells of tobacco (Nicotiana tabacum L.) BY-2 which could propagate at the same rate as the parent cells (1 mg B liter(-1)) under a lower level of boron (0.01 mg B liter(-1)) were obtained. The selected cells had swollen cell walls. In the parent cells, all the RG-II occurred as a B-RG-II complex, however, two-thirds of the RG-II occurred in a monomeric form in the selected cells.

Fructosyl amino acid oxidase (FAOD) is the enzyme catalyzing the oxidative deglycation of Amadori compounds, such as fructosyl amino acids, yielding the corresponding amino acids, glucosone, and H 2 O 2 . In a previous report, we determined the primary structures of cDNAs coding for FAODs from two fungal strains Aspergillus terreus AP1 and Penicillium janthinellum and we found that both fungal FAODs included the putative peroxisome targeting signal 1 (PTS1) at the carboxyl terminal (Yoshida, N. et al. , Eur. J. Biochem., 242, 499–505, 1996). In this study, we determined the intracellular localization of FAODs in these two fungi. Subcellular fractionation experiments and immuno-electronmicroscopic observations, together with the previous findings indicated that the FAODs were localized in peroxisomes of A. terreus AP1 and P. janthinellum . These FAODs were also found to belong to a new member of “peroxisomal sarcosine oxidase family protein” in eucaryotic cells.

A boron-polysaccharide complex was isolated and purified from germinating lily (Lilium longiflorum) pollen grains. From an analysis of sugar constituents, the sugar component of the complex is revealed to be rhamnogalacturonan II (RG-II). Immunocytochemistry using an antibody against the boron-RG-II complex of radish root cell walls demonstrated that the epitope is localized in the cell walls of elongating pollen tubes.

The mechanism of peroxisome proliferation is poorly understood. Candida boidinii is a methylotrophic yeast that undergoes rapid and massive peroxisome proliferation and serves as a good model system for this process. Pmp30A and Pmp30B (formerly designated Pmp31 and Pmp32, respectively) are two closely related proteins in a polyploid strain of this yeast that are strongly induced by diverse peroxisome proliferators such as methanol, oleate, and D-alanine. The function of these proteins is not understood. To study this issue, we used a recently described haploid strain (S2) of C. boidinii that can be manipulated genetically. We now report that strain S2 contains a single PMP30 gene very similar in sequence (greater than 93% identity at the DNA level) to PMP30A and PMP30B. When PMP30 was disrupted, cell growth on methanol was greatly inhibited, and cells grown in both methanol and oleate had fewer, larger, and more spherical peroxisomes than wild-type cells. A similar phenotype was recently described for Saccharomyces cerevisiae cultured on oleate in which PMP27, which encodes a protein of related sequence that is important for peroxisome proliferation, was disrupted. To determine whether Pmp27 is a functional homolog of Pmp30, gentle complementation was performed. PMP30A was expressed in the PMP27 disruptant of S. cerevisiae, and PMP27 was expressed in the PMP30 disruptant of C. boidinii S2. Complementation, in terms of both cell growth and organelle size, shape, and number, was successful in both directions, although reversion to a wild-type phenotype was only partial for the PMP30 disruptant. We conclude that these proteins are functional homologs and that both Pmp30 and Pmp27 have a direct role in proliferation and organelle size rather than a role in a specific peroxisomal metabolic pathway of substrate utilization.