Dan Aoki

Distribution of coniferin in freeze-fixed stem of Ginkgo biloba L. by cryo-TOF-SIMS/SEM.

To clarify the role of coniferin in planta, semi-quantitative cellular distribution of coniferin in quick-frozen Ginkgo biloba L. (ginkgo) was visualized by cryo time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. The amount and rough distribution of coniferin were confirmed through quantitative chromatography measurement using serial tangential sections of the freeze-fixed ginkgo stem. The lignification stage of the sample was estimated using microscopic observations. Coniferin distribution visualized at the transverse and radial surfaces of freeze-fixed ginkgo stem suggested that coniferin is stored in the vacuoles, and showed good agreement with the assimilation timing of coniferin to lignin in differentiating xylem. Consequently, it is suggested that coniferin is stored in the tracheid cells of differentiating xylem and is a lignin precursor.

Lignin-rich enzyme lignin (LREL), a cellulase-treated lignin-carbohydrate derived from plants, activates myeloid dendritic cells via Toll-like receptor 4 (TLR4).

Background: Although lignins are the second major biomass, their biological activities are little known. Results: Certain barley husk-derived lignin-carbohydrate fractions strongly activated dendritic cells via the receptor protein TLR4. Conclusion: Composition of the lignin-carbohydrate, especially the characteristic distribution of neutral sugars, is essential for the immunostimulatory activity. Significance: Immunostimulatory lignin-carbohydrates could be potentially used as anti-infectious agents and as vaccine adjuvants. Lignin-carbohydrates, one of the major cell wall components, are believed to be the structures that form chemical linkage between lignin and cell wall polysaccharides. Due to the molecular complexity of lignin-containing substances, their isolation and the assignment of their biological activities have so far remained a difficult task. Here, we extracted two lignin-containing carbohydrates, lignin-rich enzyme lignin (LREL) and pure enzyme lignin (PEL), from barley husk and demonstrated that they act as immune stimulators of dendritic cells (DCs), which are particularly important in linking innate and adaptive immunity. Thioacidolysis, acid hydrolysis, and mild alkali hydrolysis of both LREL and PEL revealed that their immunostimulatory activities depended on the lignin structure and/or content, neutral sugar content (especially the characteristic distribution of galactose and mannose), and presence of an ester bond. Furthermore, we showed that the immunostimulatory potency of the lignin-carbohydrate depended on its molecular weight and degree of polymerization. We also demonstrated that the LREL-induced activation of DCs was mediated via TLR4. Thus, LREL-induced increases in the expression levels of several cell surface marker proteins, production of inflammatory cytokines IL-12p40 and TNF-α, and activation and nuclear translocation of transcription factors, as was observed in the WT DCs, were completely abrogated in DCs derived from the TLR4−/− mice but not in DCs derived from the TLR2−/−, TLR7−/−, and TLR9−/− mice. We further demonstrated that LRELs isolated from other plant tissues also activated DCs. These immunostimulatory activities of lignin-carbohydrates, extracted from edible plant tissues, could have potential relevance in anti-infectious immunity and vaccine adjuvants.

ToF-SIMS Analysis of Carbonaceous Particles in the Sample Catcher of the Hayabusa Spacecraft

Three carbonaceous category 3 particles (RA-QD02-0180, RB-QD04-0037-01, and RB-QD04-0047-02) returned in the sample catcher from the Hayabusa spacecraft were analyzed by time of flight-secondary ion mass spectrometry (ToF-SIMS) to establish an analytical procedure for determination of their origins. By the different analytical schemes, the three particles gave distinct elemental and molecular ions, in which the organic carbons commonly appear to be associated with nitrogen, silicon, and/or fluorine. The particles could be debris of silicon rubber and fluorinated compounds and are therefore man-made artifacts rather than natural organic matter.

Lignification of ray parenchyma cells (RPCs) in the xylem of Phellodendron amurense Rupr.: quantitative and structural investigation by TOF-SIMS and thioacidolysis of laser microdissection cuts of RPCs

Abstract Lignification of ray parenchyma cells (RPCs) in the sapwood (sW), transition zone (TZ), and heartwood (hW) of Phellodendron amurense Rupr. has been investigated by time-of-flight secondary ion mass spectrometry (TOF-SIMS) and thioacidolysis. The results of TOF-SIMS indicate that the relative ion intensity of lignin in RPCs increased from sW to hW, while there was almost no difference in the case of axial wood fibers. The ratio of syringyl (S) to guaiacyl (G) lignin units (S/G value) in RPCs was higher than that in wood fibers. Samples containing more RPCs were prepared by laser microdissection (LMD) and analyzed by thioacidolysis, including also the subsequent Raney nickel desulfurization, and the resulting monomers and dimers were quantitatively analyzed by GC/MS. The monomer analysis shows that lignin content and S/G values in RPCs rich samples increased from sW towards hW. The dimer analysis reveals that the S-S dimers are dominant in all types of samples. The results of TOF-SIMS and thioacidolysis of LMD samples are interpreted that the lignification of RPCs progresses from the sW towards the hW and that the chemical structure of lignin in RPCs is different from that in axial elements.

Lignification of ray parenchyma cells in the xylem of Pinus densiflora. Part I: Microscopic investigation by POM, UV microscopy, and TOF-SIMS

Abstract The lignification process from sapwood (sW) to heartwood (hW) in ray parenchyma cells (Pray) of Pinus densiflora has been analyzed by means of ultraviolet (UV) microscopy, acetyl bromide (CH3COBr) lignin determination, and time-of-flight secondary ion mass spectrometry (TOF-SIMS). The cell wall layers were localized by polarized optical microscopy (POM). POM revealed that Pray have almost no secondary wall in sW and have only the outer layer of secondary wall (S1) in the transition zone (TZ) and hW. UV microscopic observations indicated that the secondary wall of Pray, which is in contact with ray tracheids (Trray), begins to lignify in sW, while the secondary wall of Pray, which is not in contact with Trray, is partially lignified in the TZ. The secondary wall of both types of Pray is completely lignified in hW. The CH3COBr lignin content in sW is slightly lower than that in hW. In the TOF-SIMS measurements, the relative intensities of the secondary ions of guaiacyl-lignin (G-lignin) in the rays in sW are significantly lower than those in hW.

Fragmentation mechanism of the phenylcoumaran-type lignin model compound by ToF-SIMS

Abstract The experiments with model compounds revealed that time-of-flight secondary ion mass spectrometry (ToF-SIMS) is able to split the common interunit linkages of lignin, except the 5-5 linkage. In a previous study, ToF-SIMS produced characteristic secondary ions with m/z 137 and 151 (C6-C1 fragments) from the phenylcoumaran-type lignin model compound, the benzofuran ring of which has a β-5 linkage and an α-O-4 linkage. However, it is still unclear whether the fragments are from ring A with the free phenolic OH as a result of the sole cleavage of the α-O-4 link by opening the furan ring or from ring B as a result of the β-5 cleavage. In this study, the phenylcoumaran-type lignin model compound and its deuterium- and/or 13C-labeled analogues were synthesized and analyzed by ToF-SIMS. It could be clarified that the β-5 linkage is not cleaved by ToF-SIMS.

Enzymatic dehydrogenative polymerization of monolignol dimers

The structure and biosynthesis of lignin are not yet fully understood, especially the step following the initial dimerization of monolignol. Liquid chromatograph mass spectrometer (LC–MS) was used to analyze the consumption rates of monolignol dimers formed by β-O-4, β-5, and β-β couplings between coniferyl alcohols in efforts to understand the activity of monolignol dimers in enzymatic dehydrogenative polymerization. We investigated the reaction kinetics in single-component and mixed-component reaction systems containing one and two species of the dimers, respectively. A difference was observed between the consumption rates of the three dimers we tested, and the consumption rate of one dimer in the single-component reaction was different from that in a mixed-component reaction. In qualitative LC–MS analyses, coniferyl alcohol oligomers were detected in the reaction products. Some monolignol tetramers were formed by 5-5 and 5-O-4 coupling between the dimers. The results of this work suggested that monolignol dimers with β-5 and β-β linkages could function as radical mediators in enzyme-catalyzed polymerization.

Lignification of ray parenchyma cells in the xylem of Pinus densiflora. Part II: Microchemical analysis by laser microdissection and thioacidolysis

Abstract The lignification of ray parenchyma cells in the sapwood (sW) and heartwood (hW) of Pinus densiflora was investigated by thioacidolysis and the subsequent Raney nickel desulfuration. The samples rich and less rich in ray parenchyma cells were prepared by laser microdissection (LMD). The whole sections burned randomly by the laser served as the controls. Guaiacyl (G) monomers were detected in all the samples, and p-hydroxyphenyl (H) monomers were detectable only in trace amounts, while syringyl (S) units were absent, as expected in softwood. In sW samples rich in ray parenchyma cells, the yields of G monomers are significantly lower than in the other samples. The various types of G-G and one G-H dimers were detected, and the β-1′, β-5′, and 5-5′ dimers were dominant. The relative distributions of lignin interunit linkages were very similar in all the samples regardless of the abundance of the ray parenchyma cells in the sW or hW tissues.

Analysis of distribution of wood extractives in Gmelina arborea by gas chromatography and time-of-flight secondary ion mass spectrometry

Abstract The aim of the present study was to investigate the extractives of Gmelina arborea stem from the sapwood (sW) to heartwood (hW) including the transition zone (tZ) between them by means of quantitative GC-MS, while the spatial distribution of four typical compounds was analysed by TOF-SIMS. The focus was on gmelinol, paulownin, 7′-O-ethyl arboreol, and β-sitosterol, which were isolated and purified from hW. The four compounds revealed a characteristic distribution pattern corresponding to their key role in biosynthesis and depending on their susceptibility to secondary reactions in the hW.

Distribution of Cell Wall Components by TOF-SIMS

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is an analytical method that has developed just within the last few decades. It is a kind of imaging mass spectrometry and supplies mass information with a submicron lateral resolution. In this chapter, the availability of TOF-SIMS measurements in the research field of woody plants is introduced. The main polymer components of the plant cell wall, cellulose, hemicellulose, and lignin are detected as fragment ions. The comprehension of the fragmentation behavior is essential to interpret the complicated mass spectra of biological samples, and the case of lignin is specifically described. Inorganics and low-molecular-weight extractives are detectable as they are. The latest topics of cryo-TOF-SIMS dealing with frozen-hydrated samples are also presented. Heterogeneous distribution of many chemical components in plants at dried or frozen-hydrated conditions might be useful information to improve our discussion another notch.