Anti-Inflammatory Effect of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Essential Oil.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) belongs to the Cupressaceae family and is native to East Asian regions. Essential oils extracted from the leaves, bark, branches, and roots of C. obtusa have both aesthetic and medicinal properties and are thus widely used. However, detailed analyses of the active ingredients of C. obtusa extract are lacking. In this study, the sabinene content in the hydro-distillation of C. obtusa leaf essential oil (COD) was analyzed using GC-MS, and the anti-inflammatory effect of COD was compared with that of pure sabinene. Cell viability was evaluated by MTT assay, and nitric oxide (NO) production was measured using Griess reagent. Relative mRNA and protein levels were analyzed using RT-qPCR and western blot, and secreted cytokines were analyzed using a cytokine array kit. The results showed that both COD and sabinene inhibited the expression of inducible nitric oxide synthase (iNOS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in lipopolysaccharide (LPS)-induced RAW 264.7 cells. COD and sabinene also reduced the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-27, IL-1 receptor antagonist (IL-1ra), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The anti-inflammatory mechanisms of COD and sabinene partially overlap, as COD was shown to inhibit MAPKs and the JAK/STAT axis, and sabinene inhibited MAPKs, thereby preventing LPS-induced macrophage activation.

Biological Activity and Component Analyses of Chamaecyparis obtusa Leaf Extract: Evaluation of Antiwrinkle and Cell Protection Effects in UVA-Irradiated Cells.

Background and Objectives: Chamaecyparis obtusa (C. obtuse) extract has been used as a folk medicinal remedy in East Asian countries to alleviate inflammation and prevent allergies. Active oxygen causes skin aging and leads to skin cell and tissue damage. Extensive research has been conducted to control active oxygen generation to prevent skin aging. We evaluated the antioxidant activity and antiwrinkle effect of C. obtusa extract to determine its potential as a cosmetic material. Materials and Methods: The antioxidant activity of a 70% ethanol extract of C. obtusa (COE 70) and a water extract of C. obtusa (COW) was determined using 2,2-diphenyl-1-picrylhydrazy (DPPH) scavenging, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) scavenging, superoxide dismutase-like activity, xanthine oxidase inhibition, and ferric-reducing antioxidant power assays. The effective concentration of the extracts was determined using the methyl thiazolyl tetrazolium assay to evaluate their toxicity. The effects of COE 70 on the production of matrix metalloproteinases (MMPs) and procollagen, and expression of activated cytokines, interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α), in UVA-irradiated fibroblasts were determined using quantitative real-time PCR. Additionally, quercitrin, amentoflavone, hinokiflavone, and myricetin concentrations in COE 70 were determined using high-pressure high-performance liquid chromatography. Results: COE 70 had higher polyphenol and flavonoid concentrations than COW and exhibited an excellent antioxidant effect. COE 70 suppressed UVA-induced fibroblast death by 21.3% at 25 µg/mL. It also increased MMP-1, MMP-3, TNF-α, and IL-6 mRNA levels at 5-25 µg/mL compared with those in control UVA-irradiated fibroblasts. Moreover, mRNA levels of collagen type I and superoxide dismutase significantly increased, indicating the antiwrinkle and anti-inflammatory effects of the extract. Among the COE 70 components, quercitrin concentration was the highest; hence, quercitrin could be an active ingredient. Conclusions: COE 70 could be used as a natural antioxidant and antiwrinkle agent.

Cloning and functional characterization of three sesquiterpene synthase genes from Chamaecyparis formosensis Matsumura.

Terpene synthase (TPS) analysis may contribute to a better understanding of terpenoids biosynthesis and the evolution of phylogenetic taxonomy. Chamaecyparis formosensis Matsumura is an endemic and valuable conifer of Taiwan. Its excellent wood quality, fragrance, and durability make it become the five precious conifers in Taiwan. In this study, three sesquiterpene synthase genes that belong to the TPS-d2 clade were isolated and characterized through in vitro reaction of recombinant protein and in vivo reaction of Escherichia coli heterologous expression system. The main product of Cf-GerA was germacrene A using GC/MS analysis, while the product of Cf-Aco and Cf-Gor were identified as acora-4(14),8-diene and (5R,6R,10S)-α-gorgonene by using NMR analysis. These are the first reported enzymes that biosynthesize acora-4(14),8-diene and (5 R,6 R,10 S)-α-gorgonene. Both sesquiterpene synthases may isomerize the farnesyl pyrophosphate substrate to nerolidyl pyrophosphate for further cyclization. Cf-Aco may catalyze 1,6-cyclization of nerolidyl cation while Cf-Gor may catalyze through an uncharged intermediate, isogermacrene A.

Transcriptome analysis of gibberellins and abscisic acid during the flooding response in Fokienia hodginsii.

Flooding is one of the main abiotic stresses suffered by plants. Plants respond to flooding stress through regulating their morphological structure, endogenous hormone biosynthesis, and genetic signaling transduction. We previously found that Fokienia hodginsii varieties originating from Gutian exhibited typical flooding tolerance traits compared to three other provenances (Yongzhou, Sanming, Nanping), expressed as increased height, longer diameter at breast height (DBH), and smaller branch angle. Herein, the changes in endogenous gibberellins (GA) and abscisic acid (ABA) contents were measured under flooding stress in F. hodginsii, and ABA was found to decrease, whereas GA increased with time. Furthermore, the GA and ABA contents of the varieties originating from Gutian and the three other provenances were measured, and the results indicated that F. hodginsii from Gutian could respond more rapidly to flooding stress. The transcriptomes of the varieties originating from Gutian and the other three provenances were compared using RNA sequencing to explore the underlying genetic mechanisms of the flood-resistant phenotypes in F. hodginsii. The results indicated that two flood-stress response genes (TRINITY_DN142_c0_g2 and TRINITY_DN7657_c0_g1) were highly related to both the ABA and GA response in F. hodginsii.

Secondary Metabolites with Antimicrobial Activities from Chamaecyparis obtusa var. formosana.

Seven new compounds, including one dimer novel skeleton, chamaecyformosanin A (1); three diterpenes, chamaecyformosanins B-D (2-4); one sesquiterpene, chamaecyformosanin E (5); and two monoterpenes, chamaecyformosanins F and G (6 and 7) were isolated from the methanol extract of the bark of Chamaecyparis obtusa var. formosana. Their structures were established by the mean of spectroscopic analysis and the comparison of NMR data with those of known analogues. Their structures were elucidated on the basis of physicochemical evidence, in-depth NMR spectroscopic analysis, and high-resolution mass spectrometry. Furthermore, the isolated compounds were subjected to an evaluation of their antimicrobial activity. Metabolites 1, 3, and 4 present antibacterial activities. It is worth mentioning that the chemical composition of the bark of C. obtusa var. formosana has never been studied in the past. This is the first time the barks from C. obtusa var. formosana were studied and two new skeleton compounds, 1 and 7, were obtained.

Drought affects the fate of non-structural carbohydrates in hinoki cypress.

Tree species that close stomata early in response to drought are likely to suffer from an imbalance between limited carbohydrate supply due to reduced photosynthesis and metabolic demand. Our objective was to clarify the dynamic responses of non-structural carbohydrates to drought in a water-saving species, the hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.). To this end, we pulse-labeled young trees with 13CO2 10 days after the beginning of the drought treatment. Trees were harvested 7 days later, early during drought progression, and 86 days later when they had suffered from a long and severe drought. The labeled carbon (C) was traced in phloem extract, in the organic matter and starch of all the organs, and in the soluble sugars (sucrose, glucose and fructose) of the most metabolically active organs (foliage, green branches and fine roots). No drought-related changes in labeled C partitioning between belowground and aboveground organs were observed. The C allocation between non-structural carbohydrates was altered early during drought progression: starch concentration was lower by half in the photosynthetic organs, while the concentration of almost all soluble sugars tended to increase. The preferential allocation of labeled C to glucose and fructose reflected an increased demand for soluble sugars for osmotic adjustment. After 3 months of a lethal drought, the concentrations of soluble sugars and starch were admittedly lower in drought-stressed trees than in the controls, but the pool of non-structural carbohydrates was far from completely depleted. However, the allocation to storage had been impaired by drought; photosynthesis and the sugar translocation rate had also been reduced by drought. Failure to maintain cell turgor through osmoregulation and to refill embolized xylem due to the depletion in soluble sugars in the roots could have resulted in tree mortality in hinoki cypress, though the total pool of carbohydrate was not completely depleted.

Discovery and characterization of diterpene synthases in Chamaecyparis formosensis Matsum. which participated in an unprecedented diterpenoid biosynthesis route in conifer.

Chamaecyparis formosensis Matsum. is an endemic and precious coniferous species of Taiwan, and is known for a high abundance of specialized metabolites, which contributes to the excellent timber durability. Several terpenoids were identified and isolated from C. formosensis wood and needles, and exhibit anti-fungal and anti-bacterial bioactivities, which may participate in plant defense against pathogens. In various identified compounds, not only cadinene and ferruginol, were identified in C. formosensis extracts but also unique diterpenoids, which include pisferal, totarol, and derivates of isoabienol. To understand the biosynthesis of these specific diterpenoids, we conducted a series of functional characterization of the C. formosensis diterpene synthases (CfdiTPSs), which participate in skeleton formation and differentiation of diterpenes. In this study, we identified eight diTPSs from C. formosensis transcriptome, and they all contain either class I or class II motif, which indicates they are all monofunctional enzymes. These candidates consist of three class II diTPSs and five class I diTPSs, and after conducting in vivo and in vitro assays, class II diTPS CfCPS1 was characterized as a (+)-copalyl diphosphate synthase ((+)-CPS), and class I diTPSs CfKSL1 could further convert (+)-copalyl diphosphate ((+)-CPP) to levopimaradiene. Meanwhile, CfKSL1 also accepted labda-13-en-8-ol diphosphate (LPP) as substrate and formed monoyl oxide. Another class I diTPS, CfKSL4, exhibits a strong enzymatic ability of isoabienol synthase, which is firstly reported in conifer. This finding provides potential participants in the biosynthesis of unique diterpenoids, and with this knowledge, we can further expand our understanding of diterpenoid metabolism in Cupressaceae and their potential role in plant defense.

Seasonal variations in emission rates and composition of terpenoids emitted from Chamaecyparis formosensis (Cupressaceae) of different ages.

Chamaecyparis formosensis (Cupressaceae) is among the most precious endemic conifers in Taiwan. Field study was conducted on seasonal variations in emission rates and compositions of terpenoids from this tree species of two different ages. A total of 21 terpenoids were detected, of which there were 13 monoterpenoids (MTs), 4 sesquiterpenoids (STs), and 4 diterpenoids (DTs). MTs dominated the emissions in both saplings and adult trees and produced more than 80% of terpene emissions. Contrasting seasonal pattern between saplings and adult trees was found. Total actual emissions from saplings were higher in cold seasons (range, 64.40 ±â€¯13.18 to 140.74 ±â€¯18.90 ng g-1 h-1) than in warm seasons (range, 55.63 ±â€¯15.84 to 63.48 ±â€¯11.85 ng g-1 h-1). Photosynthetically active radiation (PAR) was found to be the most important factor affecting terpene emissions from saplings. On the contrary, higher emissions were found in warm seasons for adult trees (range, 101.49 ±â€¯12.29 to 181.35 ±â€¯80.15 ng g-1 h-1), and the emissions were mainly in response to temperature. Some compounds in C. formosensis of both ages (e.g., ß-myrcene, α-terpinene, trans-ß-ocimene, terpinen-4-ol, α-cedrene and trans-ß-farnesene) showed comparably higher contents in cold seasons. Results presented here provide important fundamental information for better understanding of forest bathing and estimating air quality in Taiwan.

Estimation of phloem carbon translocation belowground at stand level in a hinoki cypress stand.

At stand level, carbon translocation in tree stems has to match canopy photosynthesis and carbohydrate requirements to sustain growth and the physiological activities of belowground sinks. This study applied the Hagen-Poiseuille equation to the pressure-flow hypothesis to estimate phloem carbon translocation and evaluate what percentage of canopy photosynthate can be transported belowground in a hinoki cypress (Chamaecyparis obtusa Sieb. et Zucc.) stand. An anatomical study revealed that, in contrast to sieve cell density, conductive phloem thickness and sieve cell hydraulic diameter at 1.3 m in height increased with increasing tree diameter, as did the concentration of soluble sugars in the phloem sap. At tree level, hydraulic conductivity increased by two orders of magnitude from the smallest to the largest trees in the stand, resulting in a stand-level hydraulic conductance of 1.7 × 10-15 m Pa-1 s-1. The osmotic potential of the sap extracted from the inner bark was -0.75 MPa. Assuming that phloem water potential equalled foliage water potential at predawn, the turgor pressure in the phloem at 1.3 m in height was estimated at 0.22 MPa, 0.59 MPa lower than values estimated in the foliage. With this maximal turgor pressure gradient, which would be lower during day-time when foliage water potential drops, the estimated stand-level rate of carbon translocation was 2.0 gC m-2 day-1 (30% of daily gross canopy photosynthesis), at a time of the year when aboveground growth and related respiration is thought to consume a large fraction of photosynthate, at the expense of belowground activity. Despite relying on some assumptions and approximations, this approach, when coupled with measurements of canopy photosynthesis, may further be used to provide qualitative insight into the seasonal dynamics of belowground carbon allocation.

Discovery of potential antiausterity agents from the Japanese cypress Chamaecyparis obtusa.

The chloroform extract of the Japanese cypress Chamaecyparis obtusa was found to kill PANC-1 human pancreatic cancer cells preferentially in the nutrient-deprived medium without causing toxicity in the nutrient rich condition. Phytochemical investigation on this extract led to the isolation of a new sesquiterpene (1), together with the six sesquiterpenes (2-7) and a lignan (8). The isolated compounds were tested for their preferential cytotoxicity activity against five different human pancreatic cancer cell lines [PANC-1, MIA PaCa2, CAPAN-1, PSN-1, and KLM-1] by utilizing an antiausterity strategy. Among them, α-cadinol (2) was identified as the most active constituent. α-Cadinol (2) was found to inhibit the activation of Akt/mTOR pathway, and the hyperactivation of autophagy leading to preferential PANC-1 cell death during nutrient-starvation.

Anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa on murine models of inflammation and RAW 264.7 cells.

Antimicrobial, antifungal and anti-inflammatory effects of essential oils extracted from Chamaecyparis obtusa (EOCO) have previously been reported. In the present study, the anti-inflammatory effects of EOCO were investigated in two murine models of inflammation: Carrageenan-induced paw edema and thioglycollate-induced peritonitis, and in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells. The expression levels of proinflammatory cytokines were analyzed by ELISA, the expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were determined by western blotting, and nitrite concentration was measured using Griess reagent. In mice with carrageenan-induced edema, paw thickness and the expression levels of interleukin (IL)­1ß and IL-6 in paw homogenates were significantly decreased in the EOCO (5 and 10 mg/kg) group, as compared with the control group. In mice with thioglycollate-induced peritonitis, treatment with EOCO (5 and 10 mg/kg) reduced the number of total cells and suppressed tumor necrosis factor­α (TNF­α), IL­1ß and IL­6 levels in peritoneal fluid. In addition, EOCO reduced nitric oxide, TNF­α and IL­6 production, and suppressed iNOS and COX­2 expression in LPS­stimulated RAW 264.7 cells. These results suggest that EOCO may exert anti­inflammatory effects in vivo and in vitro, and that these effects may be associated with the inhibition of inflammatory mediators. Therefore, EOCO may be considered an effective therapeutic agent for the treatment of inflammatory diseases.

Distribution of coniferin in differentiating normal and compression woods using MALDI mass spectrometric imaging coupled with osmium tetroxide vapor treatment.

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was employed to detect monolignol glucosides in differentiating normal and compression woods of two Japanese softwoods, Chamaecyparis obtusa and Cryptomeria japonica Comparison of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry collision-induced dissociation fragmentation analysis and structural time-of-flight (MALDI-TOF CID-FAST) spectra between coniferin and differentiating xylem also confirmed the presence of coniferin in differentiating xylem. However, as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF CID-FAST spectra of sucrose were similar to those of coniferin, it was difficult to distinguish the distribution of coniferin and sucrose using MALDI-MSI and collision-induced dissociation measurement only. To solve this problem, osmium tetroxide vapor was applied to sections of differentiating xylem. This vapor treatment caused peak shifts corresponding to the introduction of two hydroxyl groups to the C=C double bond in coniferin. The treatment did not cause a peak shift for sucrose, and therefore was effective in distinguishing coniferin and sucrose. Thus, it was found that MALDI-MSI combined with osmium tetroxide vapor treatment is a useful method to detect coniferin in differentiating xylem.

Estimation of the environmental effect of natural volatile organic compounds from Chamaecyparis obtusa and their effect on atopic dermatitis-like skin lesions in mice.

Aromatherapy has been suggested as an alternative therapeutic method for the treatment of atopic dermatitis (AD), eczema and other skin diseases. In the current study, the anti-atopic properties of the volatile organic compounds of Chamaecyparis obtusa (VOCCo) were examined to determine whether they are amenable for use as a pharmaceutical candidate. The alterations in histological features, serum IgE levels and mast cell infiltration following exposure to VOCCo were determined in a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model. The results of these experiments demonstrated that VOCCo inhibited the development of AD-like skin lesions by reducing the serum IgE level and mast cell infiltration into the dermal and subcutaneous layers. This was supported by screening of immune cytokine mRNAs, including interleukin (IL)-1ß and IL-6 from the skin of DNCB-treated mice. The expression of IL-1ß and IL-6 in the skin lesions of mice was dose-dependently inhibited by treatment with VOCCo. Furthermore, treatment with VOCCo resulted in the recovery of histopathological features in AD-like skin lesions. These results suggest that VOCCo may have therapeutic and preventive effects for the development of AD.

Relative deposition of xylan and 8-5'-linked lignin structure in Chamaecyparis obtusa, as revealed by double immunolabeling by using monoclonal antibodies.

MAIN CONCLUSION: Immunolabeling by using monoclonal antibodies showed that xylan deposition precedes the formation of 8-5'-linked structure of lignin in normal and compression woods of Chamaecyparis obtusa. Xylan deposition and formation of 8-5'-linked lignin structure in differentiating xylems from normal and compression woods in Chamaecyparis obtusa were examined by immunoelectron microscopy using monoclonal antibodies (LM10 or LM11) to detect xylan localization. The 8-5'-linked lignin structure was immunolocalized using KM1 antibody. Xylan and 8-5'-linked lignin double immunolabeling was performed using secondary antibodies labeled with colloidal gold particles of different diameters. In normal wood, KM1 labeling occurred in the compound middle lamella (CML) and S1 layer during S1 layer formation and increased as S2 and S3 layers formed, with labeling occurring at the outer part of the previous layer. In compression wood, mild KM1 labeling occurred in the CML and outer part of the S1 layer at the later S1 layer formation stage, with increased labeling as the S2 layer formed. Minor labeling occurred in the outer part of the S2 layer during helical cavity formation. Comparison between KM1 labeling and KMnO4 staining suggested that lignin other than 8-5'-linked structure was formed during early lignification, and the proportion of 8-5'-linked lignin structure increased at later stages of lignification in both normal and compression woods. LM10 and LM11 labeling occurred slightly earlier than KM1 labeling, suggesting that xylan deposition preceded the formation of 8-5'-linked lignin in normal and compression woods. Less labeling by KM1, LM10, and LM11 occurred in the outer part of the S2 layer in compression wood, which has abundant lignin. Thus, lignin in these parts is composed of lignin substructures other than the 8-5' linkage.

Environmental consequences of uranium atmospheric releases from fuel cycle facility: II. The atmospheric deposition of uranium and thorium on plants.

Uranium and thorium isotopes were measured in cypress leaves, wheat grains and lettuce taken in the surroundings of the uranium conversion facility of Malvési (South of France). The comparison of activity levels and activity ratios (namely (238)U/(232)Th and (230)Th/(232)Th) in plants with those in aerosols taken at this site and plants taken far from it shows that aerosols emitted by the nuclear site (uranium releases in the atmosphere by stacks and (230)Th-rich particles emitted from artificial ponds collecting radioactive waste mud) accounts for the high activities recorded in the plant samples close to the site. The atmospheric deposition process onto the plants appears to be the dominant process in plant contamination. Dry deposition velocities of airborne uranium and thorium were measured as 4.6 × 10(-3) and 5.0 × 10(-3) m s(-1), respectively.

Vertical distribution of radiocesium in coniferous forest soil after the Fukushima nuclear power plant accident.

This study deals with the description of the vertical distribution of radiocaesium ((137)Cs and (134)Cs) in a representative coniferous forest soil, investigated 10 months after the Fukushima radioactive fallout. During soil sampling, the forest floor components (understory plants, litter (Ol-) and fermented layers (Of)) were collected and treated separately. The results indicate that radiocesium is concentrated in the forest floor, and high radiocesium transfer factor observed in the undergrowth plants (3.3). This made the forest floor an active exchanging interphase for radiocesium. The raw organic layer (Ol + Of) holds 52% (5.3 kBq m(-2)) of the Fukushima-derived and 25% (0.7 kBq m(-2)) of the pre-Fukushima (137)Cs at the time of the soil sampling. Including the pre-Fukushima (137)Cs, 99% of the total soil inventory was in the upper 10 cm, in which the organic matter (OM) content was greater than 10%, suggesting the subsequent distribution most likely depends on the OM turnover. However, the small fraction of the Fukushima-derived (137)Cs at a depth of 16 cm is most likely due to the infiltration of radiocesium-circumscribed rainwater during the fallout before that selective adsorption prevails and reduces the migration of soluble (137)Cs. The values of the depth distribution parameters revealed that the distribution of the Fukushima-derived (137)Cs was somewhat rapid.

Polyhydroxy glucose functionalized silica for the dehydration of bio-ethanol distillate.

Although most of the water in a bio-ethanol fermentation broth can be removed by distillation, a small amount of water remains in the bio-ethanol distillate as the water-ethanol azeotrope. To improve the use of ethanol as a fuel, glucose-modified silica, as an adsorbent, was prepared using a facile method and applied to the dehydration of bio-ethanol distillate. The factors affecting the adsorption capacity of the adsorbent, such as the particle size, initial concentration of water in the samples, adsorption temperature and adsorbent dose, were examined by measuring the adsorption kinetics and equilibrium. The Langmuir, Freundlich and Temkin isotherms were used to evaluate the adsorption efficiency. Of these, the Freundlich and Temkin isotherms showed a good correlation with the experimental data. The Langmuir isotherm showed some deviation from the experimental results, and indicated that adsorption in this case was not a simple monolayer adsorption. The property of the adsorbent was attributed to functionalized silica with many hydroxyl groups on its surface. An examination of the separation factors of water/ethanol revealed the modified silica to have preferential selectivity for water. Compared to activated carbon and silica, glucose-modified silica exhibited higher adsorption capacity for water under the same adsorption conditions. In addition, the glucose-modified silica adsorbent exhibited a relatively constant adsorption capacity for five adsorption/desorption cycles.

A conifer ABI3-interacting protein plays important roles during key transitions of the plant life cycle.

ABI3 (for ABSCISIC ACID INSENSITIVE3), a transcription factor of the abscisic acid signal transduction pathway, plays a major role during seed development, dormancy inception, and dormancy maintenance. This protein appears to also function in meristematic and vegetative plant tissues and under certain stress conditions. We have isolated the ABI3 gene ortholog (CnABI3) from yellow cedar (Callitropsis nootkatensis) and found that it was functionally similar to other ABI3 genes of angiosperms. Here, we report that using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we have identified another protein of yellow cedar (CnAIP2; for CnABI3 INTERACTING PROTEIN2) that physically interacts with CnABI3. Functional analyses revealed that CnAIP2 plays important roles during key transitions in the plant life cycle: (1) CnAIP2 impaired seed development and reduced seed dormancy; (2) CnAIP2 promoted root development, particularly the initiation of lateral roots, and the CnAIP2 gene promoter was exquisitely auxin sensitive; and (3) CnAIP2 promoted the transition from vegetative growth to reproductive initiation (i.e. flowering). The nature of the effects of CnAIP2 on these processes and other evidence place CnAIP2 in the category of a "global" regulator, whose actions are antagonistic to those of ABI3.

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

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

The natural abundance of 15N in plant and soil-available N indicates a shift of main plant N resources to NO3(-) from NH4(+) along the N leaching gradient.

To investigate which of ammonium (NH(4)(+)) or nitrate (NO(3)(-)) is used by plants at gradient sites with different nitrogen (N) availability, we measured the natural abundance of (15)N in foliage and soil extractable N. Hinoki cypress (Chamaecyparis obtusa Endlicher) planted broadly in Japan was selected for use in this study. We estimated the source proportion of foliar N (NH(4)(+) vs. NO(3)(-)) quantitatively using mass balance equations. The results showed that C. obtusa used mainly NH(4)(+) in N-limited forests, although the dependence of C. obtusa on NO(3)(-) was greater in other NO(3)(-)-rich forests. We regarded dissolved organic N (DON) as a potential N source because a previous study demonstrated that C. obtusa can take up glycine. Thus we added DON to our mass balance equations and calculated the source proportion using an isotope-mixing model (IsoSource model). The results still showed a positive correlation between the calculated plant N proportion of NO(3)(-) and the NO(3)(-) pool size in the soil, indicating that high NO(3)(-) availability increases the reliance of C. obtusa on NO(3)(-). Our data suggest the shift of the N source for C. obtusa from NH(4)(+) to NO(3)(-) according to the relative availability of NO(3)(-). They also show the potential of the foliar delta(15)N of C. obtusa as an indicator of the N status in forest ecosystems with the help of the delta(15)N values of soil inorganic and organic N.