Arata Yoshinaga

Proton-Dependent Coniferin Transport, a Common Major Transport Event in Differentiating Xylem Tissue of Woody Plants

Differentiating xylem tissues of both hybrid poplar and Japanese cypress exhibits almost identical transport properties of coniferin, a monolignol glucoside, suggesting the involvement of a common endomembrane H+/coniferin antiport mechanism in the lignifying tissues 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.

Enzymatic saccharification of Eucalyptus bark using hydrothermal pre-treatment with carbon dioxide

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.

Ectopic Lignification in the Flax lignified bast fiber1 Mutant Stem Is Associated with Tissue-Specific Modifications in Gene Expression and Cell Wall Composition

The cell walls of flax bast fibers contain high cellulose and low lignin levels, imparting tensile strength and flexibility. To learn more about the mechanisms responsible for this type of cell wall structure, a collection of ectopic lignin mutants was identified. Characterization of the lbf1 mutant provided key information on lignification in flax that is also relevant to other plants. Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Lignification in poplar tension wood lignified cell wall layers.

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.

Immunolocalization and structural variations of xylan in differentiating earlywood tracheid cell walls of Cryptomeria japonica

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 S1 layer in the early stages of S1 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 S1 and S2 layers (S1/S2 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 S2 layer than in the inner layer, whereas xylans labeled with LM11 antibody were almost uniformly distributed in the S2 layer. In addition, the LM10 antibody showed almost no xylan labeling in the S1/S2 region, whereas the LM11 antibody revealed strong xylan labeling in the S1/S2 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.

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-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 β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S1 layer. At this stage, CW was strongly labeled in the S1 layer, whereas no label was observed in the S1 layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S1 layer of CW tracheids significantly decreased during the formation of the S2 layer. Most β-1-4-galactan labeling was present in the outer S2 region in mature CW tracheids, and was absent in the inner S2 layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-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.

Cellular distribution of coniferin in differentiating xylem of Chamaecyparis obtusa as revealed by Raman microscopy

Abstract 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).

Anatomy and lignin distribution in reaction phloem fibres of several Japanese hardwoods

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 30-45° 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 S(1) and S(2) 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 S(1) + S(2) + 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.

Immunolocalization of 8-5′ and 8-8′ linked structures of lignin in cell walls of Chamaecyparis obtusa using monoclonal antibodies

Mouse monoclonal antibodies were generated against dehydrodiconiferyl alcohol- or pinoresinol-p-aminohippuric acid (pAHA)-bovine serum albumin (BSA) conjugate as probes that specifically react with 8-5′ or 8-8′ linked structure of lignin in plant cell walls. Hybridoma clones were selected that produced antibodies that positively reacted with dehydrodiconiferyl alcohol- or pinoresinol-pAHA–BSA and negatively reacted with pAHA–BSA and guaiacylglycerol-beta-guaiacyl ether-pAHA–BSA conjugates containing 8-O-4′ linkage. Eight clones were established for each antigen and one of each clone that positively reacted with wood sections was selected. The specificity of these antibodies was examined by competitive ELISA tests using various lignin dimers with different linkages. The anti-dehydrodiconiferyl alcohol antibody reacted specifically with dehydrodiconiferyl alcohol and did not react with other model compounds containing 8-O-4′, 8-8′, or 5-5′ linkages. The anti-pinoresinol antibody reacted specifically with pinoresinol and syringaresinol and did not react with the other model compounds containing 8-O-4′, 8-5′, or 5-5′ linkages. The antibodies also did not react with dehydrodiconiferyl alcohol acetate or pinoresinol acetate, indicating that the presence of free phenolic or aliphatic hydroxyl group was an important factor in their reactivity. In sections of Japanese cypress (Chamaecyparis obtusa), labeling by the anti-dehydrodiconiferyl alcohol antibody was found in the secondary walls of phloem fibers and in the compound middle lamellae, and secondary walls of tracheids. Weak labeling by the anti-pinoresinol antibody was found in secondary walls of phloem fibers and secondary walls and compound middle lamellae of developed tracheids. These labelings show the localization of 8-5′ and 8-8′ linked structure of lignin in the cell walls.

Occurrence of xylan and mannan polysaccharides and their spatial relationship with other cell wall components in differentiating compression wood tracheids of Cryptomeria japonica

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 S1 layer at the early S1 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 S1 layer, but showed uneven distribution in the S2 layer. AGX labeling was mainly detected in the inner S2 layer after the beginning of the helical cavity formation. The outer S2 layer showed almost no labeling of low substituted AGXs. Only a very small amount of high substituted AGXs was distributed in the outer S2 layer. These patterns of AGX labeling in the S2 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 S2 layer from the early S2 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.