Anti-SARS-CoV-2 activity of microwave solvolysis lignin from woody biomass.

The global pandemic caused by the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has had profoundly detrimental effects on our society. To combat this highly pathogenic virus, we turned our attention to an abundant renewable natural aromatic polymer found in wood. Through a chemical modification of Eucalyptus and Japanese cedar wood via acidic microwave solvolysis in equivolume mixture of 2 % (w/w) aqueous H2SO4, ethylene glycol, and toluene at 190 °C. Subsequently, we separated the resulting solvolysis products through extractions with toluene, ethyl acetate, and ethanol. Among these products, the ethyl acetate extract from Eucalyptus wood (eEAE) demonstrated the highest inhibition effects against the novel SARS-CoV-2. We further divided eEAE into four fractions, and a hexane extract from the ethanol-soluble portion, termed eEAE3, exhibited the most substantial inhibitory rate at 93.0 % when tested at a concentration of 0.5 mg/mL. Analyzing eEAE3 using pyrolysis gas chromatography-mass spectrometry revealed that its primary components are derived from lignin. Additionally, 1H-13C edited-heteronuclear single quantum coherence nuclear magnetic resonance analysis showed that the solvolysis process cleaved major lignin interunit linkages. Considering the abundance and renewability of lignin, the lignin-derived anti-SARS-CoV-2 agent presents a promising potential for application in suppressing infections within our everyday environment.

Vertical gradient of needle ozone uptake within the canopy of Cryptomeria japonica.

Leaf ozone uptake through the stomata is an important index for the ozone risk assessments on trees. Stomatal conductance (gs) and ozone concentration ([O3]), determinants of the leaf ozone uptake, are known to show vertical gradients within a tree canopy. However, less is known about the within-canopy vertical gradient of leaf ozone uptake. This study was aimed to elucidate how the vertical gradient of [O3] and gs affect needle ozone uptake within a canopy of mature Cryptomeria japonica trees in a suburban forest at Tokyo, Japan. For this purpose, a multilayer gas exchange model was applied to estimate the vertical gradient of needle gs and the accumulated ozone uptake during the study period (POD1, Phytotoxic Ozone Dose above a threshold of 1 nmol m-2 s-1). In addition, we also tested several scenarios of vertical gradient of [O3] within the canopy for sensitivity analysis. The POD1 was declined from the top to the bottom of the canopy. This tendency strongly depended on the vertical gradient of gs and was hardly affected by the changes in simulated vertical reductions of the [O3]. We further assessed the photosynthesis of sunlit needles (needles absorbing both direct and diffuse light) and shaded needles (needles only absorbing diffuse light). The photosynthesis of shaded needles in the upper half of the canopy made a great contribution to the entire canopy photosynthesis. In addition, given that their POD1 was lower than that of sunlit needles, ozone may affect sunlit and shaded needles differently. We concluded that these considerations should be incorporated into modeling of the calculation of ozone uptake for mature trees to make accurate predictions of the ozone effects on trees at the canopy scale.

Downward migration of 137Cs promotes self-cleaning of forest ecosystem by reducing root uptake of Japanese cedar in Fukushima.

Approximately 70 % of the area highly 137Cs-contaminated by the Fukushima Daiichi Nuclear Power Plant accident is forested. Decontamination works in most of these forests have not progressed, and the forestry industry remains stagnant. Although the long-term dynamics of 137Cs in the forest ecosystem will be controlled by the amount of 137Cs absorbed by roots in the future, temporal changes in 137Cs of tree roots have rarely been reported. In the present study, we monitored the depth distribution of 137Cs in the soil and absorptive very fine (VF) roots of 0.5 mm or less in a Japanese cedar forest from 2011 to 2023. As a result, the 137Cs inventory in the mineral soil increased over time due to the migration from the forest canopy and litter layers, whereas that in the VF roots tended to decrease since 2020, although there was a large variation. Temporal decrease in the exchangeable 137Cs fraction with fixation and temporal increase in VF root biomass with their growth were not clearly observed, the 137Cs concentration in the VF roots at 0-2 cm decreased with the decrease in 137Cs concentration in the litter layers. Although the 137Cs concentration in the VF roots below 2 cm tended to increase with increasing 137Cs concentration in the soil at the same depth, the downward migration of 137Cs within the soil can reduce the amount of 137Cs absorbed by roots because the VF root biomass decreases exponentially with depth. In other words, 137Cs can be removed from the long-term active cycles of forest ecosystems as they migrate deeper into the soil. This natural migration process can be regarded as a "self-cleaning" of the forest ecosystem, the green and sustainable remediation using such self-cleaning should be actively adopted for the future forest management.

Discrimination of male-sterility and male-fertility in Japanese cedar (Cryptomeria japonica) using near-infrared diffuse transmission spectroscopy.

The increasing demand for pollen-free seedlings of Japanese cedar (Cryptomeria japonica) has created a need for a simple method to discriminate between male-sterile and male-fertile strobili. The objective of this study was to establish a classification model to quickly and easily distinguish male-sterile and male-fertile strobili in C. japonica using near-infrared (NIR) diffuse transmission spectroscopy. The absorbance spectra of C. japonica were obtained for three different months from December 2022 to February 2023 and preprocessed using three methods: untreated, smoothing, and second derivative. Principal component analysis was applied to the NIR spectra and classification models were built using a support vector machine. The sample collected in January 2023 showed the highest discrimination accuracy of 89.38% with the smoothing preprocessing, which was improved to 89.97% by limiting the wavelengths to the NIR region. Furthermore, discrimination accuracy for independent test data was evaluated by splitting the data into training and testing sets using January 2023 data with smoothing preprocessing. The discrimination accuracy for test data sets was more than 85%, and the misclassification ratio was less than 20% for each sample group. These results indicate the potential of using NIR diffuse transmission spectroscopy to discriminate between male-sterility and fertility in C. japonica.

Carbon stock projection for four major forest plantation species in Japan.

Carbon sequestration via afforestation and forest growth is effective for mitigating global warming. Accurate and robust information on forest growth characteristics by tree species, region, and large-scale land-use change is vital and future prediction of forest carbon stocks based on this information is of great significance. These predictions allow exploring forestry practices that maximize carbon sequestration by forests, including wood production. Forest inventories based on field measurements are considered the most accurate method for estimating forest carbon stocks. Japan's national forest inventories (NFIs) provide stand volumes for all Japanese forests, and estimates from direct field observations (m-NFIs) are the most reliable. Therefore, using the m-NFI from 2009 to 2013, we selected four major forest plantation species in Japan: Cryptomeria japonica, Chamaecyparis obtusa, Pinus spp., and Larix kaempferi and presented their forest age-carbon density function. We then estimated changes in forest carbon stocks from the past to the present using the functions. Next, we investigated the differences in the carbon sequestration potential of forests, including wood production, between five forestry practice scenarios with varying harvesting and afforestation rates, until 2061. Our results indicate that, for all four forest types, the estimates of growth rates and past forest carbon stocks in this study were higher than those considered until now. The predicted carbon sequestration from 2011 to 2061, assuming that 100 % of harvested carbon is retained for a long time, twice the rate of harvesting compared to the current rate, and a 100 % afforestation rate in harvested area, was three to four times higher than that in a scenario with no harvesting or replanting. Our results suggest that planted Japanese forests can exhibit a high carbon sequestration potential under the premise of active management, harvesting, afforestation, and prolonging the residence time of stored carbon in wood products with technology development.

Year-round dynamics of arbuscular mycorrhizal fungi communities in the roots and surrounding soils of Cryptomeria japonica.

Arbuscular mycorrhizal fungi (AMF) live simultaneously inside and outside of host plant roots for a functional mycorrhizal symbiosis. Still, the year-round dynamics and relationships between soil properties and AMF communities of trees in forest ecosystems remain unclear. We collected paired root and soil samples of the same Cryptomeria japonica trees at two forest sites (five trees at each site) every 2 months over a year. Total DNA was extracted from roots and soil separately and soil physicochemical properties were measured. With Illumina's next-generation amplicon sequencing targeting the small subunit of fungal ribosomal DNA, we clarified seasonal dynamics of soil properties and AMF communities. Soil pH and total phosphorus showed significant seasonality while total carbon, nitrogen, and C/N did not. Only pH was a good predictor of the composition and dynamics of the AMF community. The total AMF community (roots + soil) showed significant seasonality because of variation from May to September. Root and soil AMF communities were steady year-round, however, with similar species richness but contained significantly different AMF assemblages in any sampling month. Despite the weak seasonality in the communities, the top two dominant OTUs showed significant but different shifts between roots and soils across seasons with strong antagonistic relationships. In conclusion, few dominant AMF taxa are dynamically shifting between the roots and soils of C. japonica to respond to seasonal and phenological variations in their microhabitats. AMF inhabiting forest ecosystems may have high environmental plasticity to sustain a functional symbiosis regardless of seasonal variations that occur in the soil.

Effect of litter removal five years after the Fukushima accident on 137Cs uptake by Japanese cedar.

Removal of litter-associated 137Cs from the forest floor (litter removal) can reduce the 137Cs uptake by plants; however, the proposed effective period for litter removal was 1-2 years after the Fukushima Dai-ichi Nuclear Power Plant (FDNPP) accident. This is because the 137Cs in forest soil migrates rapidly from the litter to surface mineral soil layers in Japanese forests, and thus the effectiveness of litter removal will quickly become limited. However, it is unknown whether this approach can be applied to forests whose vertical migration of 137Cs in the forest soil is relatively slow. Herein, we compared the 137Cs activity concentration in the inner bark of the Japanese cedar (Cryptomeria japonica) between litter removal (conducted in September and October 2016, 5 years after the accident) and in control areas in Kawauchi Village, Fukushima Prefecture, where the vertical migration of 137Cs was relatively slow from the litter to surface mineral soil layers. Air dose rates (ambient dose equivalent) in the litter removal area were significantly lower than those in the control area in 2022, and the 137Cs inventory in the forest soil in litter removal area also tended to be lower than that in the control area. In Japanese cedars with similar levels of outer bark contamination, the 137Cs activity concentration in the inner bark in the litter removal area was significantly lower than that in the control area, and consistent trends were also found when comparing the 137Cs activity concentration in the leaves of Stephanandra incisa and Wisteria floribunda obtained from the same forest. Thus, the litter removal 5 years after the FDNPP accident may have reduced the 137Cs uptake in Japanese cedar in an evergreen coniferous forest where the vertical migration of 137Cs is relatively slow in the forest soil.

Decreased Basophil Activation against House Dust Mite after Japanese Cedar Pollen Subcutaneous Immunotherapy: A Retrospective Study.

BACKGROUND: Allergen-specific immunotherapy (AIT), an established treatment for allergic diseases, prevents the development of other allergic manifestations. Although the mechanisms remain unclear, AIT has been shown to reduce basophil activation (BA) against nontarget allergens. OBJECTIVES: The aim of this study was to assess immunological changes in Dermatophagoides farinae (Der f) after Japanese cedar pollen (JCP)-based subcutaneous immunotherapy (SCIT) monotherapy. METHOD: The data of 16 patients (age: 6-37 years) with JCP-induced allergic rhinitis who were sensitive to Der f (serum Der f-specific immunoglobulin E [IgE] level >0.34 kUA/L) and received JCP-based SCIT for 5 years were reviewed retrospectively. BA by Der f and JCP extracts and serum-specific IgE and immunoglobulin G4 (IgG4) levels against these allergens were evaluated before and after completing 5 years of JCP-based SCIT monotherapy. RESULTS: The areas under the dose-response curves of BA by Der f and JCP extracts were significantly reduced (p = 0.02 and p = 0.002, respectively). JCP-specific IgE levels decreased and JCP-specific IgG4 levels increased significantly (p < 0.001 for both), whereas Der f-specific IgE and IgG4 levels did not change significantly. CONCLUSIONS: JCP-based SCIT monotherapy reduced Der f-specific BA. These findings suggest that JCP-based SCIT has the potential to modulate immune response toward nontarget allergens.

Sublingual immunotherapy for cedar pollinosis possibly triggers eosinophilic esophagitis.

Sublingual immunotherapy (SLIT) is an effective and popular treatment for cedar pollinosis. Although SLIT can cause allergic side effects, eosinophilic esophagitis (EoE) is a lesser-known side effect of SLIT. A 26-year-old male with cedar pollinosis, wheat-dependent exercise-induced anaphylaxis, and food allergies to bananas and avocados presented with persistent throat itching, difficulty swallowing, heartburn, and anterior chest pain 8 days after starting SLIT for cedar pollinosis. Laboratory examination showed remarkably elevated eosinophils, and esophagogastroduodenoscopy revealed linear furrows in the entire esophagus. Histological examination of an esophageal biopsy specimen revealed high eosinophil levels. The patient was strongly suspected with EoE triggered by SLIT. The patient was advised to switch from the swallow to the spit method for SLIT, and the symptoms associated with SLIT-triggered EoE were reduced after switching to the spit method. This case highlights the importance of recognizing SLIT-triggered EoE as a potential side effect of SLIT for cedar pollinosis, especially with the increasing use of SLIT in clinical practice. EoE can occur within a month after initiating SLIT in patients with multiple allergic conditions, as observed in our case. Furthermore, the spit method should be recommended for patients who experience SLIT-triggered EoE before discontinuing SLIT.

Effects of forest thinning on sap flow dynamics and transpiration in a Japanese cedar forest.

Historically, forest thinning in Japan was conducted to obtain high-quality timber from plantations. Today, in contrast, thinning is also motivated by forest water balance and climate change considerations. It is in this context that the present study examines the effects of thinning on the ecophysiological responses of remaining trees, which are inadequately understood, especially in relation to changes in the magnitude and duration of transpiration. Sap flux densities were measured in both outer and inner sapwood to obtain stand-scale transpiration for two years in the pre-thinning state and three years post-thinning. The effects of thinning on transpiration were quantitatively evaluated based on canopy conductance models. The larger increases in outer sap flux density were found in the first year after the treatment, while those in inner sap flux density were detected in the second and third years. The remaining trees required a few of years to adjust to improved light conditions of the lower crown, resulting in a delayed response of inner sap flux density. As a result of this lag, transpiration was reduced to 71 % of the pre-thinning condition in the first year, but transpiration recovered to the pre-thinning levels in the second and third years due to compensating contributions from inner sap flow. In terms of more accurately chronicling the thinning effect, the distribution of sap flux density with respect to its radial pattern, is necessary. Such measurements are key to more comprehensively examining the ecophysiological response of forest plantations to thinning and, ultimately, its effect on the forest water balance.

[CLINICAL EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH CEDAR POLLEN IN A YEAR OF MASSIVE CEDAR POLLEN DISPERSAL AND ITS EFFECT ON JAPANESE CYPRESS POLLINOSIS].

BACKGROUND: We investigated the effect of sublingual immunotherapy (SLIT) on Japanese cedar and cypress pollinosis in a mass scattering year of cedar pollen (total counts: 12353 grains/cm2, approximately four times the average). METHODS: SLIT patients of 84 in the first year, 107 in the second year and 67 in the third year treated with Cedarcure® were included. During the 2023 cedar and cypress pollen dispersal season, nasal and eye symptoms and general symptoms were examined using a visual analog scale (VAS). RESULTS: The VAS during the cedar season significantly decreased to improve with the number of years of SLIT, but the VAS during the cypress period slightly increased. A questionnaire survey before SLIT revealed that only 0.9% to 3.6% of patients had strong cypress symptoms. However, sneezing (p＜0.01), rhinorrhea, itchy eyes and total symptoms (p＜0.05) in the third year of SLIT were significantly stronger during the cypress period than during the cedar period. The VAS of total symptoms during the Japanese cedar pollen season were not affected by the pollen count, and the cure rate was affected by SLIT in the third year. CONCLUSION: The clinical efficacy of cedar SLIT on cedar pollinosis was high even in a mass scattering year, and the effect increased with the number of years of treatment. On the other hand, it was shown that the clinical efficacy on cypress pollinosis was insufficient.

[A MULTICENTER CLINICAL SURVEY ABOUT THE PREVALENCE OF JAPANESE CYPRESS POLLINOSIS AND THE EFFICACY OF SUBLINGUAL IMMUNOTHERAPY WITH JAPANESE CEDAR POLLEN EXTRACT DURING JAPANESE CYPRESS POLLEN DISPERSAL PERIOD].

BACKGROUND: Little is known whether sublingual immunotherapy using Japanese cedar pollen extract (cedar SLIT) is effective for not only Japanese cedar pollinosis but also Japanese cypress pollinosis. We investigated the prevalence rate of Japanese cypress pollinosis, efficacy of cedar SLIT on cypress pollinosis and patients' wish to receive cypress SLIT. METHODS: We investigated a multi-center (31 institutions), cross-sectional survey using a self-administrated questionnaire with four questions for patients received cedar SLIT aged from 5 to 69 years old. RESULTS: 2523 subjects were enrolled for analysis. 83.4% of them had pollinosis symptoms during cypress season before cedar SLIT. In such patients, 37.4% experienced lessened efficacy of cedar SLIT during cypress season. Both the prevalence of cypress pollinosis and the lessened efficacy of cedar SLIT on cypress pollinosis were significantly seen in western Japan as compared to eastern Japan. 76.1% of the subject having cypress pollinosis before SLIT wished to receive cypress SLIT if it is available. CONCLUSION: A lessened efficacy of cedar SLIT during cypress season was broadly seen in Japan, and further showed a regional difference. Together with the finding of high wish by patients, these results suggest a development of cypress SLIT is desirable.

CRISPR/Cas9-mediated disruption of CjACOS5 confers no-pollen formation on sugi trees (Cryptomeria japonica D. Don).

Sugi (Cryptomeria japonica D. Don) is an economically important coniferous tree in Japan. However, abundant sugi pollen grains are dispersed and transported by the wind each spring and cause a severe pollen allergy syndrome (Japanese cedar pollinosis). The use of pollen-free sugi that cannot produce pollen has been thought as a countermeasure to Japanese cedar pollinosis. The sugi CjACOS5 gene is an ortholog of Arabidopsis ACOS5 and rice OsACOS12, which encode an acyl-CoA synthetase that is involved in the synthesis of sporopollenin in pollen walls. To generate pollen-free sugi, we mutated CjACOS5 using the CRISPR/Cas9 system. As a result of sugi transformation mediated by Agrobacterium tumefaciens harboring the CjACOS5-targeted CRISPR/Cas9 vector, 1 bp-deleted homo biallelic mutant lines were obtained. Chimeric mutant lines harboring both mutant and wild-type CjACOS5 genes were also generated. The homo biallelic mutant lines had no-pollen in male strobili, whereas chimeric mutant lines had male strobili with or without pollen grains. Our results suggest that CjACOS5 is essential for the production of pollen in sugi and that its disruption is useful for the generation of pollen-free sugi. In addition to conventional transgenic technology, genome editing technology, including CRISPR/Cas9, can confer new traits on sugi.

Two R2R3-MYB transcription factors from Chinese cedar (Cryptomeria fortunei Hooibrenk) are involved in the regulation of secondary cell wall formation.

As the most abundant renewable energy source, wood comprises the secondary cell wall (SCW). SCW biosynthesis involves lignin and cellulose deposition. Increasing studies have illustrated that R2R3-MYB transcription factors (TFs) play pivotal roles in affecting lignin accumulation and SCW formation. Nevertheless, the regulatory roles of R2R3-MYBs are still unresolved in Cryptomeria fortunei Hooibrenk cambium and wood formation. To dissect the potentials of CfMYBs, we successfully cloned and intensively studied the functions of CfMYB4 and CfMYB5 in SCW formation and abiotic stress response. They both contained the conserved MYB domain capable of forming a special structure that could bind to the core motifs of downstream genes. The phylogenetic tree implied that two CfMYBs clustered into different evolutionary branches. They were predominantly expressed in the stem and were localized to the nucleus. Furthermore, CfMYB4 functioned as an activator to enhance lignin and cellulose accumulation, and increase the SCW thickness by elevating the expression levels of SCW-related genes. By contrast, CfMYB5 negatively regulated lignin and cellulose biosynthesis, and decreased SCW formation by reducing the expression of SCW biosynthetic genes. Our data not only highlight the regulatory functions of CfMYBs in lignin deposition but also provide critical insights into the development of strategies for the genetic improvement of Cryptomeria fortunei wood biomass.

Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Radiocesium mobility in different parts of the two major tree species in Fukushima.

Radiocesium (137Cs) released in the Fukushima Dai-ichi Nuclear Power Plant accident is still cycling in the forest ecosystem. We examined the mobility of 137Cs in the external parts-leaves/needles, branches, and bark-of the two major tree species in Fukushima, Japanese cedar (Cryptomeria japonica) and konara oak (Quercus serrata). This variable mobility will likely lead to spatial heterogeneity of 137Cs and difficulty in predicting its dynamics for decades. We conducted leaching experiments on these samples by using ultrapure water and ammonium acetate. In Japanese cedar, the 137Cs percentage leached from current-year needles was 26-45% (ultrapure water) and 27-60% (ammonium acetate)-similar to those from old needles and branches. In konara oak, the 137Cs percentage leached from leaves was 47-72% (ultrapure water) and 70-100% (ammonium acetate)-comparable to those from current-year and old branches. Relatively poor 137Cs mobility was observed in the outer bark of Japanese cedar and in organic layer samples from both species. Comparison of the results from corresponding parts revealed greater 137Cs mobility in konara oak than in Japanese cedar. We suggest that more active cycling of 137Cs occurs in konara oak.

Paired Root-Soil Samples and Metabarcoding Reveal Taxon-Based Colonization Strategies in Arbuscular Mycorrhizal Fungi Communities in Japanese Cedar and Cypress Stands.

Arbuscular mycorrhizal fungi (AMF) in the roots and soil surrounding their hosts are typically independently investigated and little is known of the relationships between the communities of the two compartments. We simultaneously collected root and surrounding soil samples from Cryptomeria japonica (Cj) and Chamaecyparis obtusa (Co) at three environmentally different sites. Based on molecular and morphological analyses, we characterized their associated AMF communities. Cj was more densely colonized than Co and that root colonization intensity was significantly correlated with soil AMF diversity. The communities comprised 15 AMF genera dominated by Glomus and Paraglomus and 1443 operational taxonomic units (OTUs) of which 1067 and 1170 were in roots and soil, respectively. AMF communities were significantly different among sites, and the root AMF communities were significantly different from those of soil at each site. The root and soil AMF communities responded differently to soil pH. At the genus level, Glomus and Acaulospora were abundant in roots while Paraglomus and Redeckera were abundant in soil. Our findings suggest that AMF colonizing roots are protected from environmental stresses in soil. However, the root-soil-abundant taxa have adapted to both environments and represent a model AMF symbiont. This evidence of strategic exploitation of the rhizosphere by AMF supports prior hypotheses and provides insights into community ecology.