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.

Decadal trends in 137Cs concentrations in the bark and wood of trees contaminated by the Fukushima nuclear accident.

Understanding the actual situation of radiocesium (137Cs) contamination of trees caused by the Fukushima nuclear accident is essential for predicting the future contamination of wood. Particularly important is determining whether the 137Cs dynamics within forests and trees have reached apparent steady state. We conducted a monitoring survey of four major tree species (Japanese cedar, Japanese cypress, konara oak, and Japanese red pine) at multiple sites. Using a dynamic linear model, we analyzed the temporal trends in 137Cs activity concentrations in the bark (whole), outer bark, inner bark, wood (whole), sapwood, and heartwood during the 2011-2020 period. The activity concentrations were decay-corrected to September 1, 2020, to exclude the decrease due to the radioactive decay. The 137Cs concentrations in the whole and outer bark samples showed an exponential decrease in most plots but a flat trend in one plot, where 137Cs root uptake is considered to be high. The 137Cs concentration ratio (CR) of inner bark/sapwood showed a flat trend but the CR of heartwood/sapwood increased in many plots, indicating that the 137Cs dynamics reached apparent steady state within one year in the biologically active parts (inner bark and sapwood) and after several to more than 10 years in the inactive part (heartwood). The 137Cs concentration in the whole wood showed an increasing trend in six plots. In four of these plots, the increasing trend shifted to a flat or decreasing trend. Overall, the results show that the 137Cs dynamics within forests and trees have reached apparent steady state in many plots, although the amount of 137Cs root uptake in some plots is possibly still increasing 10 years after the accident. Clarifying the mechanisms and key factors determining the amount of 137Cs root uptake will be crucial for predicting wood contamination.

Mitospore formation on pure cultures of Tuber japonicum (Tuberaceae, Pezizales) in vitro.

The members of the genus Tuber are Ascomycota that form ectomycorrhizal associations with various coniferous and broadleaf tree species. In the teleomorphic stage, the species of the genus produce fruit bodies known as true truffles. Recent studies have discovered mitosporic structures, including spore mats, of several Tuber species on forest soils, indicating the presence of a cryptic anamorphic stage or an unknown reproductive strategy. Here, we report in vitro mitospore formation on the mycelium of T. japonicum, which belongs to the Japonicum clade, collected in several regions in Japan. Twenty of the 25 strains formed mitospores on modified Melin-Norkrans agar medium, indicating that mitospore formation is likely a common trait among strains of T. japonicum. The fungus forms repeatedly branched conidiophores on aerial hyphae on colonies and generates holoblastic mitospores sympodially on the terminal and near apical parts and/or occasionally on the middle and basal parts of the conidiogenous cells. Mitospores are hyaline and elliptical, obovate, oblong, or occasionally bacilliform, with a vacuole and often distinct hilar appendices. Formation of mitospores by T. japonicum in vitro is useful in understanding the functions of mitospores in the genus Tuber under controlled environmental conditions.

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.

Xylem Water Distribution in Woody Plants Visualized with a Cryo-scanning Electron Microscope.

A scanning electron microscope installed cryo-unit (cryo-SEM) allows specimen observation at subzero temperatures and has been used for exploring water distribution in plant tissues in combination with freeze fixation techniques using liquid nitrogen (LN2). For woody species, however, preparations for observing the xylem transverse-cut surface involve some difficulties due to the orientation of wood fibers. Additionally, higher tension in the water column in xylem conduits can occasionally cause artifactual changes in water distribution, especially during sample fixation and collection. In this study, we demonstrate an efficient procedure to observe the water distribution within the xylem of woody plants in situ by using a cryostat and cryo-SEM. At first, during sample collection, measuring the xylem water potential should determine whether high tension is present in the xylem conduits. When the xylem water potential is low (< ca. -0.5 MPa), a tension relaxation procedure is needed to facilitate better preservation of the water status in xylem conduits during sample freeze fixation. Next, a watertight collar is attached around the tree stem and filled with LN2 for freeze fixation of the water status of xylem. After harvesting, care should be taken to ensure that the sample is preserved frozen while completing the procedures of sample preparation for observation. A cryostat is employed to clearly expose the xylem transverse-cut surface. In cryo-SEM observations, time adjustment for freeze-etching is required to remove frost dust and accentuate the edge of the cell walls on the viewing surface. Our results demonstrate the applicability of cryo-SEM techniques for the observation of water distribution within xylem at cellular and subcellular levels. The combination of cryo-SEM with non-destructive in situ observation techniques will profoundly improve the exploration of woody plant water flow dynamics.

Dual Functions of TEMPO-Oxidized Cellulose Nanofibers in Oil-in-Water Emulsions: A Pickering Emulsifier and a Unique Dispersion Stabilizer.

The emulsifying and dispersing mechanisms of oil-in-water emulsions stabilized by 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO)-oxidized cellulose nanofibers (CNFs) have been investigated. The emulsifying mechanism was studied by changing the oil/water interfacial tension from 8.5 to 53.3 mN/m using various types of oils. The results showed that the higher the oil/water interfacial tension, the greater is the amount of CNFs adsorbed at the oil/water interface, making the CNF-adsorbed oil-in-water emulsions thermodynamically more stable. Moreover, the amount of CNFs adsorbed on the surfaces of the oil droplets increased with increasing interfacial area. The dispersion stability of the oil droplets was dominated by the CNF concentration in the water phase. Above the critical concentration (0.15% w/w), the CNFs formed network structures in the water phase, and the emulsion was effectively stabilized against creaming. Emulsion formation and the CNF network structures in the emulsion were visualized by cryo-scanning electron microscopy.

Fabrication of ultrathin nanocellulose shells on tough microparticles via an emulsion-templated colloidal assembly: towards versatile carrier materials.

Here, we develop a robust approach to forming an ∼8 nm thick cellulose nanofiber (CNF) shell on polymer microparticles through an emulsion-templated assembly. The median diameter of the CNF-shelled microparticles was 3.0 µm. The microparticles showed good dispersibility in water with a ζ-potential of -46.7 ± 0.5 mV and had good mechanical resistance. The surface CNF shells showed pH-sensitive drug loading/releasing properties, which suggest potential for a range of therapeutic and biomedical applications.

Temporal trends in 137Cs concentrations in the bark, sapwood, heartwood, and whole wood of four tree species in Japanese forests from 2011 to 2016.

To understand the changes in radiocesium (137Cs) concentrations in stem woods after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident, we investigated 137Cs concentrations in the bark, sapwood, heartwood, and whole wood of four major tree species at multiple sites with different levels of radiocesium deposition from the FDNPP accident since 2011 (since 2012 at some sites): Japanese cedar at four sites, hinoki cypress and Japanese konara oak at two sites, and Japanese red pine at one site. Our previous report on 137Cs concentrations in bark and whole wood samples collected from 2011 to 2015 suggested that temporal variations were different among sites even within the same species. In the present study, we provided data on bark and whole wood samples in 2016 and separately measured 137Cs concentrations in sapwood and heartwood samples from 2011 to 2016; we further discussed temporal trends in 137Cs concentrations in each part of tree stems, particularly those in 137Cs distributions between sapwood and heartwood, in relation to their species and site dependencies. Temporal trends in bark and whole wood samples collected from 2011 to 2016 were consistent with those reported in samples collected from 2011 to 2015. Temporal variations in 137Cs concentrations in barks showed either a decreasing trend or no clear trend, implying that 137Cs deposition in barks is inhomogeneous and that decontamination is relatively slow in some cases. Temporal trends in 137Cs concentrations in sapwood, heartwood, and whole wood were different among species and also among sites within the same species. Relatively common trends within the same species, which were increasing, were observed in cedar heartwood, and in oak sapwood and whole wood. On the other hand, the ratio of 137Cs concentration in heartwood to that in sapwood (fresh weight basis) was commonly increased to more than 2 in cedar, although distinct temporal trends were not found in the other species, for which the ratio was around 1 in cypress and pine and below 0.5 in oak, suggesting that 137Cs transfer from sapwood to heartwood shows species dependency. Consequently, the species dependency of 137Cs transfer within the tree appears easily, while that from the environment to the trees can be masked by various factors. Thus, prediction of 137Cs concentrations in stem wood should be carried out carefully as it still requires investigations at multiple sites with a larger sample size and an understanding of the species-specific 137Cs transfer mechanism.

Facile Route to Transparent, Strong, and Thermally Stable Nanocellulose/Polymer Nanocomposites from an Aqueous Pickering Emulsion.

Cellulose nanofibril (CNF) is a promising nanofiller for polymer nanocomposite materials, and a critical challenge in designing these materials is organization of the nanostructure using a facile process. Here, we report a facile aqueous preparation process for nanostructured polystyrene (PS)/CNF composites via the formation of a CNF-stabilized Pickering emulsion. PS nanoparticles, with a narrow size distribution, were synthesized by free radical polymerization in water using CNF as a stabilizer. The nanoparticles were easily collected by filtration, and the resulting material had a composite structure of PS nanoparticles embedded in a CNF framework. The PS/CNF nanocomposite showed high optical transparency, strength, and thermal dimensional stability. Thus, this technique provides a simple and environmentally friendly method for the preparation of novel CNF/polymer nanocomposite materials.

Nanocellulose-stabilized Pickering emulsions and their applications.

Pickering emulsion, which is an emulsion stabilized by solid particles, offers a wide range of potential applications because it generally provides a more stable system than surfactant-stabilized emulsion. Among various solid stabilizers, nanocellulose may open up new opportunities for future Pickering emulsions owing to its unique nanosizes, amphiphilicity, and other favorable properties (e.g. chemical stability, biodegradability, biocompatibility, and renewability). In this review, the preparation and properties of nanocellulose-stabilized Pickering emulsions are summarized. We also provide future perspectives on their applications, such as drug delivery, food, and composite materials.

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.

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.

Effect of epicuticular wax crystals on the localization of artificially deposited sub-micron carbon-based aerosols on needles of Cryptomeria japonica.

Elucidation of the mechanism of adsorption of particles suspended in the gas-phase (aerosol) to the outer surfaces of leaves provides useful information for understanding the mechanisms of the effect of aerosol particles on the growth and physiological functions of trees. In the present study, we examined the localization of artificially deposited sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica, a typical Japanese coniferous tree species, by field-emission scanning electron microscopy. The clusters (aggregates) of carbon-based particles were deposited on the needle surface regions where epicuticular wax crystals were sparsely distributed. By contrast, no clusters of the particles were found on the needle surface regions with dense distribution of epicuticular wax crystals. Number of clusters of carbon-based particles per unit area showed statistically significant differences between regions with sparse epicuticular wax crystals and those with dense epicuticular wax crystals. These results suggest that epicuticular wax crystals affect distribution of carbon-based particles on needles. Therefore, densely distributed epicuticular wax crystals might prevent the deposition of sub-micron-sized carbon-based particles on the surfaces of needles of Cryptomeria japonica to retain the function of stomata.

Cavitation of intercellular spaces is critical to establishment of hydraulic properties of compression wood of Chamaecyparis obtusa seedlings.

BACKGROUND AND AIMS: When the orientation of the stems of conifers departs from the vertical as a result of environmental influences, conifers form compression wood that results in restoration of verticality. It is well known that intercellular spaces are formed between tracheids in compression wood, but the function of these spaces remains to be clarified. In the present study, we evaluated the impact of these spaces in artificially induced compression wood in Chamaecyparis obtusa seedlings. METHODS: We monitored the presence or absence of liquid in the intercellular spaces of differentiating xylem by cryo-scanning electron microscopy. In addition, we analysed the relationship between intercellular spaces and the hydraulic properties of the compression wood. KEY RESULTS: Initially, we detected small intercellular spaces with liquid in regions in which the profiles of tracheids were not rounded in transverse surfaces, indicating that the intercellular spaces had originally contained no gases. In the regions where tracheids had formed secondary walls, we found that some intercellular spaces had lost their liquid. Cavitation of intercellular spaces would affect hydraulic conductivity as a consequence of the induction of cavitation in neighbouring tracheids. CONCLUSIONS: Our observations suggest that cavitation of intercellular spaces is the critical event that affects not only the functions of intercellular spaces but also the hydraulic properties of compression wood.

Characteristics of initial deposition and behavior of radiocesium in forest ecosystems of different locations and species affected by the Fukushima Daiichi Nuclear Power Plant accident.

After the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident, information about stand-level spatial patterns of radiocesium initially deposited in the surrounding forests was essential for predicting the future dynamics of radiocesium and suggesting a management plan for contaminated forests. In the first summer (approximately 6 months after the accident), we separately estimated the amounts of radiocesium ((134)Cs and (137)Cs; Bq m(-2)) in the major components (trees, organic layers, and soils) in forests of three sites with different contamination levels. For a Japanese cedar (Cryptomeria japonica) forest studied at each of the three sites, the radiocesium concentration greatly differed among the components, with the needle and organic layer having the highest concentrations. For these cedar forests, the proportion of the (137)Cs stock in the aboveground tree biomass varied from 22% to 44% of the total (137)Cs stock; it was 44% in highly contaminated sites (7.0 × 10(5) Bq m(-2)) but reduced to 22% in less contaminated sites (1.1 × 10(4) Bq m(-2)). In the intermediate contaminated site (5.0-5.8 × 10(4) Bq m(-2)), 34% of radiocesium was observed in the aboveground tree biomass of the Japanese cedar stand. However, this proportion was considerably smaller (18-19%) in the nearby mixed forests of the Japanese red pine (Pinus densiflora) and deciduous broad-leaved trees. Non-negligible amounts of (134)Cs and (137)Cs were detected in both the sapwood and heartwood of all the studied tree species. This finding suggested that the uptake or translocation of radiocesium had already started within 6 months after the accident. The belowground compartments were mostly present in the organic layer and the uppermost (0-5 cm deep) mineral soil layer at all the study sites. We discussed the initial transfer process of radiocesium deposited in the forest and inferred that the type of initial deposition (i.e., dry versus wet radiocesium deposition), the amount of rainfall after the accident, and the leaf biomass by the tree species may influence differences in the spatial pattern of radiocesium by study plots. The results of the present study and further studies of the spatial pattern of radiocesium are important for modeling future radiocesium distribution in contaminated forest ecosystems.

Recovery of Physiological Traits in Saplings of Invasive Bischofia Tree Compared with Three Species Native to the Bonin Islands under Successive Drought and Irrigation Cycles.

Partial leaf shedding induced by hydraulic failure under prolonged drought can prevent excess water consumption, resulting in delayed recovery of carbon productivity following rainfall. To understand the manner of water use of invasive species in oceanic island forests under a fluctuating water regime, leaf shedding, multiple physiological traits, and the progress of embolism in the stem xylem under repeated drought-irrigation cycles were examined in the potted saplings of an invasive species, Bischofia javanica Blume, and three endemic native species, Schima mertensiana (Sieb. Et Zucc,) Koitz., Hibiscus glaber Matsum, and Distylium lepidotum Nakai, from the Bonin Islands, Japan. The progress of xylem embolism was observed by cryo-scanning electron microscopy. The samples exhibited different processes of water saving and drought tolerance based on the different combinations of partial leaf shedding involved in embolized conduits following repeated de-rehydration. Predawn leaf water potential largely decreased with each successive drought-irrigation cycle for all tree species, except for B. javanica. B. javanica shed leaves conspicuously under drought and showed responsive stomatal conductance to VPD, which contributed to recover leaf gas exchange in the remaining leaves, following a restored water supply. In contrast, native tree species did not completely recover photosynthetic rates during the repeated drought-irrigation cycles. H. glaber and D. lepidotum preserved water in vessels and adjusted leaf osmotic rates but did not actively shed leaves. S. mertensiana exhibited partial leaf shedding during the first cycle with an osmotic adjustment, but they showed less responsive stomatal conductance to VPD. Our data indicate that invasive B. javanica saplings can effectively use water supplied suddenly under drought conditions. We predict that fluctuating precipitation in the future may change tree distributions even in mesic or moist sites in the Bonin Islands.

Seasonal variation in formation, structure, and chemical properties of phloem in Picea abies as studied by novel microtechniques.

MAIN CONCLUSION: Phloem production and structural development were interlinked with seasonal variation in the primary and secondary metabolites of phloem. Novel microtechniques provided new perspectives on understanding phloem structure and chemistry. To gain new insights into phloem formation in Norway spruce (Picea abies), we monitored phloem cell production and seasonal variation in the primary and secondary metabolites of inner bark (non-structural carbohydrates and phenolic stilbene glucosides) during the 2012 growing season in southern and northern Finland. The structure of developing phloem was visualised in 3D by synchrotron X-ray microtomography. The chemical features of developing phloem tissues isolated by laser microdissection were analysed by chemical microanalysis. Within-year phloem formation was associated with seasonal changes in non-structural carbohydrates and phenolic extractive contents of inner bark. The onset of phloem cell production occurred in early and mid-May in southern and northern Finland, respectively. The maximal rate of phloem production and formation of a tangential band of axial phloem parenchyma occurred in mid-June, when total non-structural carbohydrates peaked (due to the high amount of starch). In contrast, soluble sugar content dropped during the most active growth period and increased in late summer and winter. The 3D visualisation showed that the new axial parenchyma clearly enlarged from June to August. Sub-cellular changes appeared to be associated with accumulation of stilbene glucosides and soluble sugars in the newest phloem. Stilbene glucosides also increased in inner bark during late summer and winter. Our findings may indicate that stilbene biosynthesis in older phloem predominantly occurs after the formation of the new band(s) of axial parenchyma. The complementary use of novel microtechniques provides new perspectives on the formation, structure, and chemistry of phloem.

The accumulation pattern of ferruginol in the heartwood-forming Cryptomeria japonica xylem as determined by time-of-flight secondary ion mass spectrometry and quantity analysis.

BACKGROUND AND AIMS: Heartwood formation is a unique phenomenon of tree species. Although the accumulation of heartwood substances is a well-known feature of the process, the accumulation mechanism remains unclear. The aim of this study was to determine the accumulation process of ferruginol, a predominant heartwood substance of Cryptomeria japonica, in heartwood-forming xylem. METHODS: The radial accumulation pattern of ferruginol was examined from sapwood and through the intermediate wood to the heartwood by direct mapping using time-of-flight secondary ion mass spectrometry (TOF-SIMS). The data were compared with quantitative results obtained from a novel method of gas chromatography analysis using laser microdissection sampling and with water distribution obtained from cryo-scanning electron microscopy. KEY RESULTS: Ferruginol initially accumulated in the middle of the intermediate wood, in the earlywood near the annual ring boundary. It accumulated throughout the entire earlywood in the inner intermediate wood, and in both the earlywood and the latewood in the heartwood. The process of ferruginol accumulation continued for more than eight annual rings. Ferruginol concentration peaked at the border between the intermediate wood and heartwood, while the concentration was less in the latewood compared with the earlywood in each annual ring. Ferruginol tended to accumulate around the ray parenchyma cells. In addition, at the border between the intermediate wood and heartwood, the accumulation was higher in areas without water than in areas with water. CONCLUSIONS: TOF-SIMS clearly revealed ferruginol distribution at the cellular level. Ferruginol accumulation begins in the middle of intermediate wood, initially in the earlywood near the annual ring boundary, then throughout the entire earlywood, and finally across to the whole annual ring in the heartwood. The heterogeneous timing of ferruginol accumulation could be related to the distribution of ray parenchyma cells and/or water in the heartwood-forming xylem.