Color variation in young and senescent leaves of Formosan sweet gum (Liquidambar formosana) by the gene regulation of anthocyanidin biosynthesis.

In certain plants, leaf coloration occurs in young and senescent leaves; however, it is unclear whether these two developmental stages are controlled by the same regulatory mechanisms. Formosan sweet gum (Liquidambar formosana Hance) is a subtropical deciduous tree species that possesses attractive autumnal leaf coloration. The color of young leaves is closer to purplish red, while senescent leaves are more orange-red to dark red. It was confirmed that delphinidin and cyanidin are the two anthocyanidins that contribute to the color of Formosan sweet gum leaves, and the content of different anthocyanins influences the appearance of color. To elucidate the regulation of anthocyanidin biosynthesis, recombinant DIHYDROFLAVONOL-4-REDUCTASEs (LfDFR1 and LfDFR2) (EC 1.1.1.234) were produced, and their substrate acceptability was investigated both in vitro and in planta. The functions of flavanones and dihydroflavonols modification by FLAVONOID 3' HYDROXYLASE (LfF3'H1) (EC 1.14.14.82) and FLAVONOID 3'5' HYDROXYLASE (LfF3'5'H) (EC 1.14.14.81) were verified using a transient overexpression experiment in Nicotiana benthamiana. The results showed that LfMYB5 induced LfF3'5'H and LfMYB123 induced both LfF3'H1 and LfDFR1 in spring when the leaves were expanding, whereas LfMYB113 induced LfF3'H1, LfDFR1, and LfDFR2 in late autumn to winter when the leaves were undergoing leaf senescence. In conclusion, the color variation of Formosan sweet gum in young and senescent leaves was attributed to the composition of anthocyanidins through the transcriptional regulation of LfF3'H1 and LfF3'5'H by LfMYB5, LfMYB113, and LfMYB123.

Biochemical characterization of diterpene synthases of Taiwania cryptomerioides expands the known functional space of specialized diterpene metabolism in gymnosperms.

Taiwania cryptomerioides is a monotypic gymnosperm species, valued for the high decay resistance of its wood. This durability has been attributed to the abundance of terpenoids, especially the major diterpenoid metabolite ferruginol, with antifungal and antitermite activity. Specialized diterpenoid metabolism in gymnosperms primarily recruits bifunctional class-I/II diterpene synthases (diTPSs), whereas monofunctional class-II and class-I enzymes operate in angiosperms. In this study, we identified a previously unrecognized group of monofunctional diTPSs in T. cryptomerioides, which suggests a distinct evolutionary divergence of the diTPS family in this species. Specifically, five monofunctional diTPS functions not previously observed in gymnosperms were characterized, including monofunctional class-II enzymes forming labda-13-en-8-ol diphosphate (LPP, TcCPS2) and (+)-copalyl diphosphate (CPP, TcCPS4), and three class-I diTPSs producing biformene (TcKSL1), levopimaradiene (TcKSL3) and phyllocladanol (TcKSL5), respectively. Methyl jasmonate (MeJA) elicited the accumulation of levopimaradiene and the corresponding biosynthetic diTPS genes, TcCPS4 and TcKSL3, is consistent with a possible role in plant defense. Furthermore, TcCPS4 and TcKSL3 are likely to contribute to abietatriene biosynthesis via levopimaradiene as an intermediate in ferruginol biosynthesis in Taiwania. In conclusion, this study provides deeper insight into the functional landscape and molecular evolution of specialized diterpenoid metabolism in gymnosperms as a basis to better understand the role of these metabolites in tree chemical defense.

Identification, Functional Characterization, and Seasonal Expression Patterns of Five Sesquiterpene Synthases in Liquidambar formosana.

Terpenoids are a large group of important secondary metabolites that are involved in a variety of physiological mechanisms, and many are used commercially in the cosmetics and pharmaceutical industries. During the past decade, the topic of seasonal variation in terpenoid biosynthesis has garnered increasing attention. Formosan sweet gum ( Liquidambar formosana Hance) is a deciduous tree species. The expression of terpene synthase and accumulation of terpenoids in leaves may vary in different seasons. Here, four sesquiterpene synthases (i.e., LfTPS01, LfTPS02, LfTPS03, and LfTPS04) and a bifunctional mono/sesquiterpene synthase ( LfTPS05) were identified from Formosan sweet gum. The gene expression of LfTPS01, LfTPS02, and LfTPS03 showed seasonal diversification, and, in addition, expression of LfTPS04 and LfTPS05 was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, trans-ß-caryophyllene, α-copaene/δ-cadinene, and nerolidol/linalool, respectively. The data indicated that the sesquiterpenoid content in the essential oil of Formosan sweet gum leaves shows seasonal differences that were correlated to the sesquiterpene synthase gene expression.

A R2R3-MYB Gene LfMYB113 is Responsible for Autumn Leaf Coloration in Formosan sweet gum (Liquidambar formosana Hance).

The regulation of autumn leaf coloration in deciduous trees has long been an enigma. Due to the fact that different coloration phenotypes may be considered when planting, more understanding of the regulation mechanism is needed. In this study, a R2R3-MYB transcription factor gene LfMYB113 was identified from a subtropical deciduous tree species Formosan sweet gum (Liquidambar formosana Hance). The expression patterns of LfMYB113 in four selected phenotypes were different and were positively correlated with leaf anthocyanin content. In a 35S::LfMYB113 transgenic Nicotiana tabacum plant, both the early and late genes in the anthocyanin biosynthetic pathway were shown to be up-regulated. It was also shown that LfMYB113 can activate the promoter sequence of LfDFR1 and LfDFR2. Transient overexpression of LfMYB113 in Nicotiana benthamiana showed strong anthocyanin accumulation and pre-senescence; the latter was confirmed by up-regulation of senescence-associated genes. In addition, the activation of proLfSGR::YFP by LfMYB113 in transient experiments indicated that LfMYB113 may have a role in regulation of Chl degradation. To our knowledge, this is the first time a R2R3-MYB transcription factor has been functionally identified as one of the key regulators of autumn leaf coloration and autumn leaf senescence.

Genomic and transcriptomic analyses of the medicinal fungus Antrodia cinnamomea for its metabolite biosynthesis and sexual development.

Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over $100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.

Integrating RNA-seq and ChIP-seq data to characterize long non-coding RNAs in Drosophila melanogaster.

BACKGROUND: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs. RESULTS: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not. CONCLUSIONS: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.

Metabolite Profiling and Comparison of Bioactivity in Antrodia cinnamomea and Antrodia salmonea Fruiting Bodies.

Antrodia cinnamomea is a precious edible mushroom endemic to Taiwan that has been claimed to have significant health promotion activities. Antrodia salmonea is a new species of the genus Antrodia. In this study, we compared the metabolites and bioactivity of A. cinnamomea and A. salmonea fruiting bodies. The volatiles of A. cinnamomea and A. salmonea were characterized and 3,4,5-trimethoxybenzaldehyde was found to be the most abundant compound in A. cinnamomea; the other abundant compounds were δ-guaiene, isolongifolene, 1-octen-3-ol, 4-terpinenol, α-guaiene, and p-cymene. In A. salmonea, the main volatiles were α-cedrene, 1-octen-3-ol, D-limonene, cadinadiene, germacrene D, isolongifolene, and α-muurolene. Furthermore, five ergostane-type triterpenoids and two lanostane-type triterpenoids were selected as index compounds characterizing A. cinnamomea and A. salmonea extracts. The content of each compound varied between the two species. (R,S)-antcin B was the most abundant compound in A. cinnamomea fruiting bodies (75.18 ± 0.11 µg/mg). However, (R,S)-antcin C (184.85 ± 0.96 µg/mg) was the major triterpenoid in the A. salmonea fruiting body. Furthermore, two compounds, antcin M and methyl antcinate K, were only present in the A. salmonea fingerprint; therefore, antcin M and methyl antcinate K may be important for distinguishing between A. cinnamomea and A. salmonea fruiting bodies. Finally, examination of anti-inflammation activity and cytotoxicity showed that A. salmonea had more anti-inflammatory activity than A. cinnamomea; however, A. salmonea was more cytotoxic than A. cinnamomea. In conclusion, the composition and bioactivity of the fruiting bodies of A. cinnamomea and A. salmonea varies. Therefore, it is recommended that further toxicological evaluation and investigation of the biological activity of A. salmonea is carried out to ensure its safe and efficacious use as an alternative to A. cinnamomea.

Transcriptome analysis of a subtropical deciduous tree: autumn leaf senescence gene expression profile of Formosan gum.

Autumn leaf senescence is a spectacular natural phenomenon; however, the regulation networks controlling autumnal colors and the leaf senescence program remain largely unelucidated. Whether regulation of leaf senescence is similar in subtropical deciduous plants and temperate deciduous plants is also unknown. In this study, the gene expression of a subtropical deciduous tree, Formosan gum (Liquidambar formosana Hance), was profiled. The transcriptomes of April leaves (green leaves, 'G') and December leaves (red leaves, 'R') were investigated by next-generation gene sequencing. Out of 58,402 de novo assembled contigs, 32,637 were annotated as putative genes. Furthermore, the L. formosana-specific microarray designed based on total contigs was used to extend the observation period throughout the growing seasons of 2011-2013. Network analysis from the gene expression profile focused on the genes up-regulated when autumn leaf senescence occurred. LfWRKY70, LfWRKY75, LfWRKY65, LfNAC1, LfSPL14, LfNAC100 and LfMYB113 were shown to be key regulators of leaf senescnece, and the genes regulated by LfWRKY75, LfNAC1 and LfMYB113 are candidates to link chlorophyll degradation and anthocyanin biosynthesis to senescence. In summary, the gene expression profiles over the entire year of the developing leaf from subtropical deciduous trees were used for in silico analysis and the putative gene regulation in autumn coloration and leaf senescence is discussed in this study.

Characterization of the 2,3-Oxidosqualene Cyclase Gene from Antrodia cinnamomea and Enhancement of Cytotoxic Triterpenoid Compound Production.

Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.

Functional identification of specialized diterpene synthases from Chamaecyparis obtusa and C. obtusa var. formosana to illustrate the putative evolution of diterpene synthases in Cupressaceae.

Chamaecyparis obtusa and C. obtusa var. formosana of the Cupressaceae family are well known for their fragrance and excellent physical properties. To investigate the biosynthesis of unique diterpenoid compounds, diterpene synthase genes for specialized metabolite synthesis were cloned from C. obtusa and C. obtusa var. formosana. Using an Escherichia coli co-expression system, eight diterpene synthases (diTPSs) were characterized. CoCPS and CovfCPS are class II monofunctional (+)-copalyl diphosphate synthases [(+)-CPSs]. Class I monofunctional CoLS and CovfLS convert (+)-copalyl diphosphate [(+)-CPP] to levopimaradiene, CoBRS, CovfBRS1, and CovfBRS3 convert (+)-CPP to (-)-beyerene, and CovfSDS converts (+)-CPP to (-)-sandaracopimaradiene. These enzymes are all monofunctional diterpene syntheses in Cupressaceae family of gymnosperm, and differ from those in Pinaceae. The discovery of the enzyme responsible for the biosynthesis of tetracyclic diterpene (-)-beyerene was characterized for the first time. Diterpene synthases with different catalytic functions exist in closely related species within the Cupressaceae family, indicating that this group of monofunctional diterpene synthases is particularly prone to the evolution of new functions and development of species-specific specialized diterpenoid constituents.

Antifungal Activity of Cedrol from Cunninghamia lanceolate var. konishii against Phellinus noxius and Its Mechanism.

Phellinus noxius is a highly destructive fungus that causes brown root disease in trees, leading to decay and death. In Taiwan, five prized woods-Taiwania cryptomerioides, Calocedrus macrolepis var. formosana, Cunninghamia lanceolata var. konishii, Chamaecyparis formosensis, and Chamaecyparis obtusa var. formosana-are known for their fragrance and durability. This study aims to explore the anti-brown-root-rot-fungus activity of Cunninghamia lanceolata var. konishii (CL) essential oil (CLOL) and its primary components, while also delving into their mechanisms of action and inhibition pathways. The essential oil (CLOL) from CL wood demonstrated significant efficacy against P. noxius, with an inhibitory concentration (IC50) of 37.5 µg/mL. Cedrol, the major component (78.48%) in CLOL, emerged as a potent antifungal agent, surpassing the reference drug triflumizole. Further assays with cedrol revealed a stronger anti-brown-root-disease activity (IC50 = 15.7 µg/mL) than triflumizole (IC50 = 32.1 µg/mL). Scanning electron microscopy showed deformation and rupture of fungal hyphae treated with CLOL and cedrol, indicating damage to the fungal cell membrane. Cedrol-induced oxidative stress in P. noxius was evidenced by increased reactive oxygen species (ROS) levels, leading to DNA fragmentation, mitochondrial membrane potential reduction, and fungal apoptosis through the mitochondrial pathway. Gel electrophoresis confirmed cedrol-induced DNA fragmentation, whereas TUNEL staining demonstrated increased apoptosis with rising cedrol concentrations. Moreover, protein expression analysis revealed cedrol-triggered release of cytochrome c, activation of caspase-9, and subsequent caspase-3 activation, initiating a caspase cascade reaction. This groundbreaking study establishes cedrol as the first compound to induce apoptosis in P. noxius while inhibiting its growth through oxidative stress, an increase in mitochondrial membrane permeability, and activation of the mitochondrial pathway. The findings offer compelling evidence for cedrol's potential as an effective antifungal agent against the destructive brown root disease caused by P. noxius.

Savinin Triggers Programmed Cell Death of Ray Parenchyma Cells in Heartwood Formation of Taiwania cryptomerioides Hayata.

The purpose of this study was to investigate the relationship between lignan biosynthesis and programmed cell death (PCD) of ray parenchyma cells during the heartwood formation of Taiwania (Taiwania cryptomerioides Hayata). Since the PCD of ray parenchyma cells and the synthesis of lignans are the two main processes involved in the formation of heartwood, both of which need to be completed through gene regulation. Based on the results of genomics and bioinformatics analysis, that the PCD of tracheids are induced by genotoxic, and the PCD of ray parenchyma cells is induced by biological factors, such as fungi, bacteria, and viruses, which could induce oxidative stress. According to the results of time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis, lignans are produced in ray parenchyma cells, and the accumulation of savinin and its downstream lignans might be the cause of PCD in ray parenchyma cells. An in vitro experiment further confirmed that the accumulation of savinin could cause protoplasts of Taiwania's xylem to produce taiwanin A, which is the marker of heartwood formation in Taiwania. Resulting in an increase in reactive oxygen species (ROS) content, which could induce oxidative stress in ray parenchyma cells and potentially lead to PCD. Based on these findings, we conclude that accumulation of savinin could be induced PCD of ray parenchyma cells in heartwood formation in Taiwania.

Monoterpene synthases contribute to the volatile production in tana (Zanthoxylum ailanthoides) through indigenous cultivation practices.

Tana (Zanthoxylum ailanthoides), a perennial deciduous species in the Rutaceae family, possesses leaves with a unique fragrance that indigenous peoples incorporate into their traditional cuisine. In Kalibuan, the cultivated tana trees were pruned repeatedly to maintain a shorter height, which led to the growth of new leaves that were spicier and pricklier. Tana leaves contain a range of volatile terpenoids, and the pungent aroma may arise from the presence of monoterpenoids. To gain insight into the biosynthetic pathway, five candidate monoterpene synthase genes were cloned and characterized using a purified recombinant protein assay. The main product of Za_mTPS1, Za_mTPS2, and Za_mTPS5 is sabinene, geraniol, and (E)-ß-ocimene, respectively. The main product of Za_mTPS3 and Za_mTPS4 is linalool. Real-time PCR analysis revealed that Za_mTPS1 and Za_mTPS5 are expressed at higher levels in prickly leaves of cultivated tana, suggesting that they may contribute to the distinctive aroma of this plant.

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.

Anti-Inflammatory Activities of Constituents from Cinnamomum insularimontanum Hayata Leaves and Their Mechanisms.

Cinnamomum insularimontanum is an endemic species of Taiwan. Although most Cinnamomum plants have significant biological activity, the bioactivity investment of C. insularimontanum is rare. Since inflammation plays an important role in many diseases, anti-inflammatory compounds can be developed into healthcare products. Therefore, we first conducted a study on the anti-inflammatory activity of C. insularimontanum leaves. First, we examined the antiinflammation activity of essential oil from C. insularimontanum leaves, and it revealed potent anti-inflammatory activity. A total of 23 volatile compounds were identified in C. insularimontanum leaves' essential oil by using GC/MS analysis. Among them were 1,8-cineole (35.94%), α-eudesmol (6.17%), pinene (7.55%), sabinene (5.06%), and isobornyl acetate (4.81%). According to previous studies, 1,8-cineole might be an anti-inflammation principal compound of C. insularimontanum leaves. Next, the ethanolic extracts of C. insularimontanum leaves also exhibited good anti-inflammatory activity. Two bioactive compounds, isoburmanol (F1) and burmanol (F2), were isolated from the ethyl acetate soluble fraction by using the bioactivity-guided separation protocol and spectroscopic analysis. F1 was obtained from C. insularimontanum for the first time, and F2 was isolated for the first time from natural resources. Both F1 and F2 could inhibit the production of nitric oxide (NO), and the IC50 values were 14.0 µM and 43.8 µM, RAW 264.7 cells after induction of lipopolysaccharide. Furthermore, F1 and F2 also revealed significant inhabitation effects on iNOS and COX-2 protein expression. The anti-inflammation activity of F1 and F2 was different from the common pathway of inhibiting NF-κB. Both of them could inhibit the production of NO and PGE2 by directly inhibiting the AP-1 (c-Jun) protein and then inhibiting the downstream iNOS and COX-2. Although both F1 and F2 possessed significant anti-inflammatory activity, the activity of F1 was better than F2. Through molecular docking simulation analysis, the results show that F1 and F2 interact with AP-1, inhibit the binding of AP-1 to DNA, and cause AP-1 to fail to transcribe the related factors of inflammation. The binding ability of AP-1 and F1 was stronger than F2, and that is the reason why F1 exhibited better activities in both downstream proteins and inflammatory cytokines. Based on the results obtained in this study, the essential oil and F1 and F2 isolated from C. insularimontanum leaves have good anti-inflammatory activities, and it is expected to be used as a reference for the development of medical care products in the future.

SSR individual identification system construction and population genetics analysis for Chamaecyparis formosensis.

Chamaecyparis formosensis is an endemic species of Taiwan, threatened from intensive use and illegal felling. An individual identification system for C. formosensis is required to provide scientific evidence for court use and deter illegal felling. In this study, 36 polymorphic simple sequence repeat markers were developed. By applying up to 28 non-linked of the developed markers, it is calculated that the cumulative random probability of identity (CPI) is as low as 1.652 × 10-12, and the identifiable population size is up to 60 million, which is greater than the known C. formosensis population size in Taiwan. Biogeographical analysis data show that C. formosensis from four geographic areas belong to the same genetic population, which can be further divided into three clusters: SY (Eastern Taiwan), HV and GW (Northwestern Taiwan), and MM (Southwestern Taiwan). The developed system was applied to assess the provenance of samples with 88.44% accuracy rate and therefore can serve as a prescreening tool to reduce the range required for comparison. The system developed in this study is a potential crime-fighting tool against illegal felling.

Inhibitory effect of human breast cancer cell proliferation via p21-mediated G1 cell cycle arrest by araliadiol isolated from Aralia cordata Thunb.

A new polyacetylenic compound, araliadiol, was isolated from the leaves of Aralia cordata Thunb. (Araliaceae). The structure of araliadiol was determined to be 3( S),8( R)-pentadeca-1,9( Z)-diene-4,6-diyne-3,8-diol by MS, NMR, IR, and UV spectroscopic analysis as well as Mosher ester reaction. Araliadiol displayed a significant inhibitory effect on the growth of a human breast adenocarcinoma cell line (MCF-7), with an IC (50) value for cytotoxicity of 6.41 µg/mL. Cell cycle analysis revealed that the proportion of cells in the G (1) phase of the cell cycle increased in a dose-dependent manner (from 54.7 % to 72.0 %) after 48 h exposure to araliadiol at dosages ranging from 0 to 80 µM. The results suggest that araliadiol inhibits cell cycle progression of MCF-7 at the G (1)-S transition. After treatment with araliadiol, phosphorylation of retinoblastoma protein (Rb) in MCF-7 cells was inhibited, accompanied by a decrease in the levels of cyclin D (3) and cyclin-dependent kinase 4 (cdk4) and an increase in the expression of p21 (WAF-1/Cip1). However, the expression of phosphorylated p53 (Ser15) and Chk2 was not altered in MCF-7 cells. These findings indicate that araliadiol exhibits its growth-inhibitory effects on MCF-7 cells through downregulation of cdk4 and cyclin D (3), and upregulation of p21 (WAF-1/Cip1) by a p53-independent mechanism.

Proteomic analysis of differently cultured endemic medicinal mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou from Taiwan.

Antrodia cinnamomea is peculiar to Taiwan. It only grows on one host and is highly valued as an important component of several traditional Chinese medicines. In this study, the different protein expression profiles of artificially cultivated vegetative mycelium and wild-type basidiomatal fruiting bodies were compared and unique protein spots from wild-type basidiomatal fruiting body were investigated using 2D polyacrylamide gel electrophoresis and LC-MS/MS protein identification. Most of the wild-type proteins not seen in the artificially cultivated mycelium were associated to function in metabolism, cell stress, ROS scavenging, and cell growth. Several proteins from wild-type basidiomes, such as catalase, aryl-alcohol dehydrogenase, S-adenosyl-L-homocysteine hydrolase, intradiol dioxygenase, haloacid dyhydrogenase, alpha- and beta-form tubulin, prohibitin, septin, chaperone, and HSP90 ATPase, showed higher expression than those from artificially cultured mycelium at the mRNA level.

Cytochrome P450 genes in medicinal mushroom Antrodia cinnamomea T.T. Chang et W.N. Chou (higher Basidiomycetes) are strongly expressed during fruiting body formation.

Medicinal mushroom Antrodia cinnamomea is a higher Basidiomycetes endemic to Taiwan, where it is commonly used as a traditional folk medicine. It is well known for its multiple biologic activities and its potential for commercial development. Here, ten full lengths of cytochrome P450 (CYP) genes (ac-1 to ac-10) from A. cinnamomea were cloned and identified. With the exception of ac-3 and ac-8, which will probably be assigned as new CYP families, these genes had more than 40% amino acid identity and close evolutionary relationships to known CYPs. Among the ten genes, only Ac-7 did not possess a transmembrane domain but had an N-terminal signal peptide, so it was considered a novel extracellular CYP. The ten A. cinnamomea CYPs had different expression profiles in different growth conditions. In general, they were strongly expressed during the formation of fruiting bodies, especially in natural basidiomycetes. The expression of six CYPs of A. cinnamomea (ac-1 to ac-3 and ac-5 to ac-7) were strictly inhibited in the mycelia cell type. It was therefore concluded that these CYPs are most active in the fruiting bodies of A. cinnamomea.

Unveiling Monoterpene Biosynthesis in Taiwania cryptomerioides via Functional Characterization.

Taiwania cryptomerioides is a monotypic species, and its terpenoid-rich property has been reported in recent years. To uncover monoterpene biosynthesis in T. cryptomerioides, this study used transcriptome mining to identify candidates with tentative monoterpene synthase activity. Along with the phylogenetic analysis and in vitro assay, two geraniol synthases (TcTPS13 and TcTPS14), a linalool synthase (TcTPS15), and a ß-pinene synthase (TcTPS16), were functionally characterized. Via the comparison of catalytic residues, the Cys/Ser at region 1 might be crucial in determining the formation of α-pinene or ß-pinene. In addition, the Cupressaceae monoterpene synthases were phylogenetically clustered together; they are unique and different from those of published conifer species. In summary, this study aimed to uncover the ambiguous monoterpenoid network in T. cryptomerioide, which would expand the landscape of monoterpene biosynthesis in Cupressaceae species.