Bonding of Lignin and Coniferyl Alcohol by a Redox Shuttle of Low-Molecular-Weight Lignols in Enzymatic Oxidative Dehydrogenative Polymerization.

Lignin is an aromatic polymer that constitutes plant cell walls. The polymerization of lignin proceeds by radical coupling, and this process requires radicalization of the phenolic end of lignin by enzymes. However, due to the steric hindrance between enzymes, lignin, and polysaccharides, the direct oxidation of the phenolic end of lignin by the enzyme would be difficult, and the details of the growth of lignin are still unknown. In this study, enzymatic dehydrogenative polymerization experiments were conducted using coniferyl alcohol (CA) and the deuterium-labeled lignin model compound (D-LM) under a noncontact condition in which horseradish peroxidase cannot directly oxidize D-LM due to separation by a dialysis membrane. Analysis of deuterium-labeled degraded compounds obtained by a combination of methylation and thioacidolysis revealed the formation of the bond between the phenolic end of D-LM and CA, suggesting that membrane-permeable, low-molecular-weight lignols functioned as a redox shuttle mediator.

Polycyclic aromatic hydrocarbons in samples of Ryugu formed in the interstellar medium.

Polycyclic aromatic hydrocarbons (PAHs) contain ≲20% of the carbon in the interstellar medium. They are potentially produced in circumstellar environments (at temperatures ≳1000 kelvin), by reactions within cold (~10 kelvin) interstellar clouds, or by processing of carbon-rich dust grains. We report isotopic properties of PAHs extracted from samples of the asteroid Ryugu and the meteorite Murchison. The doubly-13C substituted compositions (Δ2×13C values) of the PAHs naphthalene, fluoranthene, and pyrene are 9 to 51‰ higher than values expected for a stochastic distribution of isotopes. The Δ2×13C values are higher than expected if the PAHs formed in a circumstellar environment, but consistent with formation in the interstellar medium. By contrast, the PAHs phenanthrene and anthracene in Ryugu samples have Δ2×13C values consistent with formation by higher-temperature reactions.

A lignin-derived material improves plant nutrient bioavailability and growth through its metal chelating capacity.

The lignocellulosic biorefinery industry can be an important contributor to achieving global carbon net zero goals. However, low valorization of the waste lignin severely limits the sustainability of biorefineries. Using a hydrothermal reaction, we have converted sulfuric acid lignin (SAL) into a water-soluble hydrothermal SAL (HSAL). Here, we show the improvement of HSAL on plant nutrient bioavailability and growth through its metal chelating capacity. We characterize HSAL's high ratio of phenolic hydroxyl groups to methoxy groups and its capacity to chelate metal ions. Application of HSAL significantly promotes root length and plant growth of both monocot and dicot plant species due to improving nutrient bioavailability. The HSAL-mediated increase in iron bioavailability is comparable to the well-known metal chelator ethylenediaminetetraacetic acid. Therefore, HSAL promises to be a sustainable nutrient chelator to provide an attractive avenue for sustainable utilization of the waste lignin from the biorefinery industry.

Microscopic distribution of alkaloids in freeze-fixed stems of Phellodendron amurense.

Introduction: Phellodendron amurense Rupr. contains rich alkaloids, which have been extensively applied in clinical treatments for their various biological activities. However, detailed microscopic distribution and roles of such alkaloids in P. amurense stem still need to be clarified. Methods: In this study, the distribution of eight alkaloids in the transverse surface of freeze-fixed P. amurense stems in fall and summer has been visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM), which was found in living tissues with relative contents of different alkaloids varying with the position. In addition, the contents of these alkaloids quantified by high-performance liquid chromatography (HPLC) analysis suggested the seasonal variation from fall to the following summer. Results and discussion: Distribution of eight alkaloids in the freeze-fixed stems of P. amurense from fall and summer seasons has been visualized and assigned into specific living tissues, with relative contents varying in different positions with seasons, which suggested their possible roles in the physiological processes of the plant itself or plant responding to changes in the surrounding conditions. Conclusion: This study provided a significant basis for further discussion of the genes or enzymes involved in these processes, which will contribute to investigating biosynthetic pathways and specific in planta roles of alkaloids.

Inspired by nature: Fiber networks functionalized with tannic acid and condensed tannin-rich extracts of Norway spruce bark show antimicrobial efficacy.

This study demonstrated the antibacterial and antiviral potential of condensed tannins and tannic acid when incorporated into fiber networks tested for functional material purposes. Condensed tannins were extracted from industrial bark of Norway spruce by using pressurized hot water extraction (PHWE), followed by purification of extracts by using XADHP7 treatment to obtain sugar-free extract. The chemical composition of the extracts was analyzed by using HPLC, GC‒MS and UHPLC after thiolytic degradation. The test matrices, i.e., lignocellulosic handsheets, were produced and impregnated with tannin-rich extracts, and tannic acid was used as a commercial reference. The antibacterial and antiviral efficacy of the handsheets were analyzed by using bioluminescent bacterial strains (Staphylococcus aureus RN4220+pAT19 and Escherichia coli K12+pCGLS11) and Enterovirus coxsackievirus B3. Potential bonding of the tannin-rich extract and tannic acid within the fiber matrices was studied by using FTIR-ATR spectroscopy. The deposition characteristics (distribution and accumulation patterns) of tannin compounds and extracts within fiber networks were measured and visualized by direct chemical mapping using time-of-flight secondary ion mass spectrometry (ToF-SIMS) and digital microscopy. Our results demonstrated for the first time, how tannin-rich extracts obtained from spruce bark side streams with green chemistry possess antiviral and antibacterial properties when immobilized into fiber matrices to create substitutes for plastic hygienic products, personal protection materials such as surgical face masks, or food packaging materials to prolong the shelf life of foodstuffs and prevent the spread of infections. However, more research is needed to further develop this proof-of-concept to ensure stable chemical bonding in product prototypes with specific chemistry.

Soluble organic molecules in samples of the carbonaceous asteroid (162173) Ryugu.

The Hayabusa2 spacecraft collected samples from the surface of the carbonaceous near-Earth asteroid (162173) Ryugu and brought them to Earth. The samples were expected to contain organic molecules, which record processes that occurred in the early Solar System. We analyzed organic molecules extracted from the Ryugu surface samples. We identified a variety of molecules containing the atoms CHNOS, formed by methylation, hydration, hydroxylation, and sulfurization reactions. Amino acids, aliphatic amines, carboxylic acids, polycyclic aromatic hydrocarbons, and nitrogen-heterocyclic compounds were detected, which had properties consistent with an abiotic origin. These compounds likely arose from an aqueous reaction on Ryugu's parent body and are similar to the organics in Ivuna-type meteorites. These molecules can survive on the surfaces of asteroids and be transported throughout the Solar System.

Distribution of lignans and lignan mono/diglucosides within Ginkgo biloba L. stem.

To investigate the biosynthetic pathways and regulatory mechanisms of lignans in plants, the actual distributions of lignans and lignan glucosides in flash-frozen stems of Ginkgo biloba L. (Ginkgoaceae) were studied using cryo time-of-flight secondary ion mass spectrometry coupled with scanning electron microscopy (cryo-TOF-SIMS/SEM). Four lignans and four lignan glucosides were successfully characterized. Quantitative HPLC measurements were conducted on serial tangential sections of freeze-fixed ginkgo stem to determine the amount and approximate distribution of lignan and lignan glucosides. (-)-Olivil 4,4'-di-O-ß-d-glucopyranoside (olivil DG) was the most abundant lignan glucoside in ginkgo and was distributed mainly in the phloem, ray parenchyma cells, and pith. The comparative accumulation of olivil DG revealed its possible transport pathways and storage sites in ginkgo. Although not all relevant enzymes have been identified, understanding the distributions of lignan and lignan glucosides in ginkgo stems provides significant insight into their biological functions.

Nutritional resources of the yeast symbiont cultivated by the lizard beetle Doubledaya bucculenta in bamboos.

Symbiotic fungi of wood-inhabiting insects are often considered to aid wood digestion of host insects when the associated fungi can assimilate wood-associated indigestible materials. In most cases, however, the components of wood that are utilized by fungal symbionts remain poorly understood. The lizard beetle Doubledaya bucculenta (Coleoptera, Erotylidae, Languriinae) farms the symbiotic yeast Wickerhamomyces anomalus inside the cavity of host bamboo internodes, which serves as food for larvae. To determine the carbon sources of the internodes serving as nutritional substrates for W. anomalus, we used ion exchange chromatography measurements to analyze free and structural sugar compositions in fresh pith (FP), yeast-cultured pith (YP), and larva-reared pith (LP) of internodes. Glucose and fructose were the major free sugars in FP and markedly decreased in YP and LP. For structural sugars, no sugar significantly decreased in YP or LP compared with FP. Carbon assimilation tests showed that W. anomalus assimilated glucose, mannose, fructose, and sucrose strongly, xylose and cellobiose moderately, and xylan weakly. Elemental analysis revealed that the compositions of carbon, hydrogen, and nitrogen were not significantly different among tissue types. These results suggest that W. anomalus does not consume bamboo-associated indigestible sugars but most free sugars, mainly glucose and fructose, in the pith. Our findings suggest that a symbiont's abilities may not always benefit its host in nature.

Combinations of the Aromatic Rings in ß-1 Structure Formation of Lignin Based on Quantitative Analysis by Thioacidolysis.

The amount of ß-1 structures in lignin is small; however, they are assumed to significantly affect the reactivity of lignin because they form dienone structures. A method employing thioacidolysis and subsequent desulfurization yields products that can be analyzed via gas chromatography-mass spectrometry (GC-MS) to quantify these ß-1 structures. However, the retention times and response factors of the reaction products have not been accurately determined thus far. Here, 12 standard compounds combined with p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units were synthesized, and their retention times and response factors were determined through GC-MS, using selective ions. Based on these data, we also investigated the ß-1 structures of lignocellulosic lignin samples. Our results clarified that the successful formation of the ß-1 structure was dependent on the type of aromatic rings present; there were very few ß-1 structures containing H units; and the amount of G-G type was higher and that of the heterotype, i.e., G-S type, was lower than the stochastic value.

Localization of (+)-Catechin in Picea abies Phloem: Responses to Wounding and Fungal Inoculation.

To understand the positional and temporal defense mechanisms of coniferous tree bark at the tissue and cellular levels, the phloem topochemistry and structural properties were examined after artificially induced bark defense reactions. Wounding and fungal inoculation with Endoconidiophora polonica of spruce bark were carried out, and phloem tissues were frequently collected to follow the temporal and spatial progress of chemical and structural responses. The changes in (+)-catechin, (-)-epicatechin, stilbene glucoside, and resin acid distribution, and accumulation patterns within the phloem, were mapped using time-of-flight secondary ion mass spectrometry (cryo-ToF-SIMS), alongside detailed structural (LM, TEM, SEM) and quantitative chemical microanalyses of the tissues. Our results show that axial phloem parenchyma cells of Norway spruce contain (+)-catechins, the amount of which locally increases in response to fungal inoculation. The preformed, constitutive distribution and accumulation patterns of (+)-catechins closely follow those of stilbene glucosides. Phloem phenolics are not translocated but form a layered defense barrier with oleoresin compounds in response to pathogen attack. Our results suggest that axial phloem parenchyma cells are the primary location for (+)-catechin storage and synthesis in Norway spruce phloem. Chemical mapping of bark defensive metabolites by cryo-ToF-SIMS, in addition to structural and chemical microanalyses of the defense reactions, can provide novel information on the local amplitudes and localizations of chemical and structural defense mechanisms and pathogen-host interactions of trees.

Identification of Naturally Occurring Polyamines as Root-Knot Nematode Attractants.

Root-knot nematodes (RKNs; genus Meloidogyne) are a class of plant parasites that infect the roots of many plant species. It is believed that RKNs target certain signaling molecules derived from plants to locate their hosts; however, currently, no plant compound has been unambiguously identified as a universal RKN attractant. To address this question, we screened a chemical library of synthetic compounds for Meloidogyne incognita attractants. The breakdown product of aminopropylamino-anthraquinone, 1,3-diaminopropane, as well as its related compounds, putrescine and cadaverine, were found to attract M. incognita. After examining various polyamines, M. incognita were found to be attracted specifically by natural compounds that possess three to five methylene groups between two terminal amino groups. Using cryo-TOF-SIMS/SEM, cadaverine was indeed detected in soybean root cortex cells and the surrounding rhizosphere, establishing a chemical gradient. In addition to cadaverine, putrescine and 1,3-diaminopropane were also detected in root exudate by HPLC-MS/MS. Furthermore, exogenously applied cadaverine is sufficient to enhance M. incognita infection of Arabidopsis seedlings. These results suggest that M. incognita is likely attracted by polyamines to locate the appropriate host plants, and the naturally occurring polyamines have potential applications in agriculture in developing protection strategies for crops from RKN infection.

Microscopic distribution of syringin in freeze-fixed Syringa vulgaris stems.

Monolignols are precursors of lignin, and their glucosides are often found in plants. Glucosylation creates water-soluble and chemically stable monolignols by protecting the phenolic hydroxyl group. To discuss the role of sinapyl alcohol glucoside, syringin, in planta, the cellular distribution of syringin in the transverse and radial surfaces of quick-frozen stems of Syringa vulgaris L. (lilac) was visualized by cryo-time-of-flight secondary ion mass spectrometry and scanning electron microscopy (cryo-TOF-SIMS/SEM) analyses. The amount and rough distribution of syringin were confirmed by high-performance liquid chromatography measurements using serial tangential sections of freeze-fixed lilac stems. The syringin distribution was also discussed with reference to the tissue classification from microscopic observations. Syringin was mainly found in the phloem region. In the xylem region, syringin was evenly distributed irrespective of the cell type from the cambial zone to the early differentiating stage region and selectively distributed in vessels in the later differentiating stage region. After the lignification of wood fibers, syringin was found in rays and some vessels in the initial part of the annual rings. Previously, artificially administered isotope-labeled syringin was shown to be assimilated into lignin in the differentiating xylem region. Based on this, our present data showing syringin storage in the differentiating xylem region and its variation depending on the lignification stage suggest that syringin works as a lignin precursor. Additionally, detection of syringin in vessels and rays indicates intercellular transportation of syringin in lilac stems.

Unexpected polymerization mechanism of dilignol in the lignin growing.

Lignin is an essential component of higher plants, which is built by the enzymatic dehydrogenative polymerization of monolignols. First, monolignol is enzymatically oxidized to produce the phenoxy radical, which can form resonance hybrids. Two radical resonant hybrids are coupled with each other to yield dilignol with various linkage types, of which the main structures are ß-O-4' (I), ß-5' (II) and ß-ß' (III). However, the reaction mechanism behind the addition lignol radicals to dilignol is not yet fully understood. Here, we show an unexpected reaction with structure II during enzymatic dehydrogenative polymerization, which involves cleavage of a covalent linkage and creation of a new radical coupling site. This implied that the ß-5 dilignol diversifies the growing pattern of lignin. This discovery elucidates a novel mechanism in lignin polymerization.

Direct mapping of hydrangea blue-complex in sepal tissues of Hydrangea macrophylla.

The original sepal color of Hydrangea macrophylla is blue, although it is well known that sepal color easily changes from blue through purple to red. All the colors are due to a unique anthocyanin, 3-O-glucosyldelphinidin, and both aluminum ion (Al3+) and copigments, 5-O-caffeoyl and/or 5-O-p-coumaroylquinic acid are essential for blue coloration. A mixture of 3-O-glucosyldelphinidin, 5-O-acylquinic acid, and Al3+ in a buffer solution at pH 4 produces a stable blue solution with visible absorption and circular dichroism spectra identical to those of the sepals, then, we named this blue pigment as 'hydrangea blue-complex'. The hydrangea blue-complex consists of 3-O-glucosyldelphinidin, Al3+, and 5-O-acylquinic acid in a ratio 1:1:1 as determined by the electrospray ionization time-of-flight mass spectrometry and nuclear magnetic resonance spectra. To map the distribution of hydrangea blue-complex in sepal tissues, we carried out cryo-time-of-flight secondary ion mass spectrometry analysis. The spectrum of the reproduced hydrangea blue-complex with negative mode-detection gave a molecular ion at m/z = 841, which was consistent with the results of ESI-TOF MS. The same molecular ion peak at m/z = 841 was detected in freeze-fixed blue sepal-tissue. In sepal tissues, the blue cells were located in the second layer and the mass spectrometry imaging of the ion attributable to hydrangea blue-complex overlapped with the same area of the blue cells. In colorless epidermal cells, atomic ion of Al3+ was hardly detected and potassium adduct ion of 5-O-caffeoyl and/or 3-O-acylquinic acid were found. This is the first report about the distribution of aluminum, potassium, hydrangea blue-complex, and copigment in sepal tissues and the first evidence that aluminum and hydrangea blue-complex exist in blue sepal cells and are involved in blue coloration.

Sexual and temporal variations in floral scent in the subdioecious shrub Eurya japonica Thunb.

In many flowering plants, floral scents are a significant trait for visitors, playing an important role in attracting pollinators and/or detracting herbivores. The evolution of flowering plants from hermaphroditism to dioecy is often accompanied by sexual dimorphism in floral scent. In this study, floral scents emitted by different sexual morphs of the subdioecious shrub Eurya japonica Thunb. were collected using a dynamic headspace method, and sexual and temporal variations were evaluated by gas chromatography-mass spectrometry (GC-MS). Two volatiles, α-pinene and linalool, were identified as the major components of floral scents in females, hermaphrodites, and males. The males emit higher amounts of floral scents, particularly α-pinene, compared to females or hermaphrodites. Floral scents emitted by males generally decrease as flowers enter senescence, whereas those from females or hermaphrodites do not significantly differ. Intraspecific variations in floral scents of subdioecious species provided by this study would contribute to better understanding of sexual dimorphism in floral scent.

Visualization of solute diffusion into cell walls in solution-impregnated wood under varying relative humidity using time-of-flight secondary ion mass spectrometry.

The purpose of the present study is to clarify the diffusion of non-volatile substances into cell walls during the conditioning procedure under varying relative humidities (RH). In this paper, wood blocks were impregnated using an aqueous solution of melamine formaldehyde (MF), and they were subsequently conditioned under RHs of 11, 43, and 75%. The solute that diffused into the cell walls was visualized using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The volumetric relative swelling of the samples during the conditioning procedure was calculated. The results showed increased cell wall swelling at higher RH, which may have been caused by higher MF diffusion into the cell walls and/or higher moisture content. Cryo-TOF-SIMS measurements showed that more cell cavities were unfilled with MF at higher RH, indicating that most of the MF diffused from the cell cavities into the cell walls. The relative intensity of MF in the cell walls of the cured samples was evaluated from dry-TOF-SIMS images, which showed a higher relative intensity of MF in the cell walls at higher RH. With the ability to visualize and semi-quantitatively evaluate the solute in cell walls, TOF-SIMS will serve as a powerful tool for future studies of solute diffusion mechanisms in solution-impregnated wood.

Distribution of salicifoline in freeze-fixed stems of Magnolia kobus as observed by cryo-TOF-SIMS.

Alkaloids are basic nitrogen-containing chemicals that have important physiological and pharmacological characteristics. Many vascular plant species contain alkaloids, and their roles in planta are of interest. However, the detailed distribution of alkaloids remains unclear because of their low water solubility and low concentrations in plants. In this study, we visualized the distribution of salicifoline, a water-soluble quaternary ammonium alkaloid, in the freeze-fixed stems of Magnolia kobus by cryo time-of-flight secondary ion mass spectrometry. Most of the salicifoline was distributed in living phloem tissues. In the xylem, salicifoline was detected in ray cells, lignifying wood fibres, and in vessels in the latest annual ring. The salicifoline distribution in the xylem varied with the cell wall formation stage. These results provide new insights into the storage, transportation, and role of the alkaloid salicifoline in M. kobus.

Topochemical Analysis of Cell Wall Components by TOF-SIMS.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a recently developing analytical tool and a type of imaging mass spectrometry. TOF-SIMS provides mass spectral information with a lateral resolution on the order of submicrons, with widespread applicability. Sometimes, it is described as a surface analysis method without the requirement for sample pretreatment; however, several points need to be taken into account for the complete utilization of the capabilities of TOF-SIMS. In this chapter, we introduce methods for TOF-SIMS sample treatments, as well as basic knowledge of wood samples TOF-SIMS spectral and image data analysis.

Translocation of 133Cs administered to Cryptomeria japonica wood.

To reveal the in planta behaviour of caesium (Cs), the stable isotope 133Cs was administered into 3-year-old Cryptomeria japonica seedlings by the application of 133CsCl aqueous solution to the bark surface. The administered 133Cs was quantified by ICP-MS measurements, which showed transportation of 133Cs in an ascending direction in the stem. Distribution of 133Cs was visualized using freeze-fixed C. japonica woody stem samples and cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM) analysis. Cryo-TOF-SIMS/SEM visualization suggested that 133Cs was rapidly transported radially by ray parenchyma cells followed by axial transportation by pith and axial parenchyma cells. Adsorption experiments using powdered C. japonica wood samples and X-ray absorption fine structure (XAFS) analysis suggested that 133Cs was in the hydrated state following its deposition into tracheid cell walls.

In Planta Localization of Stilbenes within Picea abies Phloem.

Phenolic stilbene glucosides (astringin, isorhapontin, and piceid) and their aglycons commonly accumulate in the phloem of Norway spruce (Picea abies). However, current knowledge about the localization and accumulation of stilbenes within plant tissues and cells remains limited. Here, we used an innovative combination of novel microanalytical techniques to evaluate stilbenes in a frozen-hydrated condition (i.e. in planta) and a freeze-dried condition across phloem tissues. Semiquantitative time-of-flight secondary ion-mass spectrometry imaging in planta revealed that stilbenes were localized in axial parenchyma cells. Quantitative gas chromatography analysis showed the highest stilbene content in the middle of collapsed phloem with decreases toward the outer phloem. The same trend was detected for soluble sugar and water contents. The specimen water content may affect stilbene composition; the glucoside-to-aglycon ratio decreased slightly with decreases in water content. Phloem chemistry was correlated with three-dimensional structures of phloem as analyzed by microtomography. The outer phloem was characterized by a high volume of empty parenchyma, reduced ray volume, and a large number of axial parenchyma with porous vacuolar contents. Increasing porosity from the inner to the outer phloem was related to decreasing compactness of stilbenes and possible secondary oxidation or polymerization. Our results indicate that aging-dependent changes in phloem may reduce cell functioning, which affects the capacity of the phloem to store water and sugar, and may reduce the defense potential of stilbenes in the axial parenchyma. Our results highlight the power of using a combination of techniques to evaluate tissue- and cell-level mechanisms involved in plant secondary metabolite formation and metabolism.