Genome-wide identification of glycoside hydrolase family 1 members reveals GeBGL1 and GeBGL9 for degrading gastrodin in Gastrodia elata.

KEY MESSAGE: We revealed the intrinsic transformation molecular mechanism of gastrodin by two ß-d-glucosidases (GeBGL1 and GeBGL9) during the processing of Gastrodia elata. Gastrodia elata is a plant resource with medicinal and edible functions, and its active ingredient is gastrodin. However, the intrinsic transformation molecular mechanism of gastrodin in G. elata has not been verified. We speculated that ß-d-glucosidase (BGL) may be the key enzymes hydrolyzing gastrodin. Here, we identified 11 GeBGL genes in the G. elata genome. These genes were unevenly distributed on seven chromosomes. These GeBGL proteins possessed motifs necessary for catalysis, namely, TF(I/M/L)N(T)E(Q)P and I(V/L)T(H/S)ENG(S). These GeBGLs were divided into five subgroups together with homologous genes from Arabidopsis thaliana, rice, and maize. Quantitative real-time PCR analysis showed GeBGL genes expression was tissue-specific. Gene cloning results showed two mutation sites in the GeBGL1 gene compared with the reference genome. And, the GeBGL4 gene has two indel fragments, which resulted in premature termination of translation and seemed to turn into a pseudogene. Furthermore, protein expression and enzyme activity results proved that GeBGL1 and GeBGL9 have the activity of hydrolyzing gastrodin into 4-hydroxybenzyl alcohol. This study revealed the function of ß-d-glucosidase in degrading active compounds during the G. elata processing for medicinal purposes. These results offer a theoretical foundation for elevating the standard and enhancing the quality of G. elata production.

[Effects of soil relative water content on growth characteristics of Armillaria spp. and seedling production of Gastrodia elata f. glauca].

The seedling survival rate, yield, and individual weight of Gastrodia elata is closely related to the soil relative water content(RWC) and the growth characteristics of the associated fungi Armillaria spp. This study explored the effects of the soil RWC on the growth characteristics of Armillaria spp. and the seedling production of G. elata f. glauca, aiming to provide guidance for breeding G. elata f. glauca and selecting elite strains of Armillaria. According to the growth characteristics on the medium for activation, thirty strains of Armillaria were classified into 4 clusters. Two strains with good growth indicators were selected from each cluster and cultiva-ted with immature tuber(Mima) and the branches of the broad-leaved trees in a water-controlled box. The results showed that the Armillaria clusters with uniaxial branches of rhizoid cords, such as clusters Ⅲ and Ⅳ, were excellent clusters in symbiosis with G. elata f. glauca. The soil RWC had significant effects on the growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca. The growth characteristics of Armillaria strains and the seedling survival rate, yield, and individual weight of G. elata f. glauca in the case of the soil RWC being 75% were significantly better than those in the case of other soil RWC. Cultivating Mima with elite strains of Armillaria, together with branches of broad-leaved trees, in the greenhouses with the artificial control of the soil RWC, can achieve efficient seedling production and Mima utilization of G. elata f. glauca.

Phialocetones A-J, C12 lactones from the rhizospheric soil-derived fungus Phialocephala sp. YUD18001 associated with Gastrodia elata.

Ten undescribed C12 polyketide phialocetones A-J, featuring twelve-, six- and five-membered lactone moieties, were isolated from a rhizospheric soil-derived Phialocephala sp. YUD18001 associated with Gastrodia elata. Their structures were established by NMR spectroscopic analysis and HRMS, while their absolute configurations were determined by computational methods and chemical reactions. All isolated compounds were evaluated for their anti-inflammatory and cytotoxic activities. As a result, phialocetone D exhibited moderate effects against NO production in lipopolysaccharide (LPS)-induced RAW264.7 cells with an IC50 value of 14.77 µM, while phialocetone E showed cytotoxicity against HL-60 and SW480 cell lines with IC50 values of 19.04 and 10.22 µM, respectively.

Para-Hydroxybenzyl Alcohol Delays the Progression of Neurodegenerative Diseases in Models of Caenorhabditis elegans through Activating Multiple Cellular Protective Pathways.

The traditional Chinese medicine Gastrodia elata (commonly called "Tianma" in Chinese) has been widely used in the treatment of rheumatism, epilepsy, paralysis, headache, and dizziness. Phenolic compounds, such as gastrodin, para-hydroxybenzyl alcohol (HBA), p-hydroxybenzaldehyde, and vanillin are the main bioactive components isolated from Gastrodia elata. These compounds not only are structurally related but also share similar pharmacological activities, such as antioxidative and anti-inflammatory activities, and effects on the treatment of aging-related diseases. Here, we investigated the effect of para-hydroxybenzyl alcohol (HBA) on neurodegenerative diseases and aging in models of Caenorhabditis elegans (C. elegans). Our results showed that HBA effectively delayed the progression of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and Huntington's disease in models of C. elegans. In addition, HBA could increase the average lifespan of N2 worms by more than 25% and significantly improve the age-related physiological functions of worms. Moreover, HBA improved the survival rate of worms under stresses of oxidation, heat, and pathogenic bacteria. Further mechanistic investigation revealed that HBA could activate FOXO/DAF-16 and SKN-1 to regulate antioxidative and xenobiotic metabolism pathway. HBA could also activate HSF-1 to regulate proteostasis maintenance pathway, mitochondrial unfolded stress response, endoplasmic stress response and autophagy pathways. The above results suggest that HBA activated multiple cellular protective pathways to increase stress resistance and protect against aging and aging-related diseases. Overall, our study indicates that HBA is a potential candidate for future development of antiaging pharmaceutical application.

Gastrodia elata Blume Polysaccharides Attenuate Vincristine-Evoked Neuropathic Pain through the Inhibition of Neuroinflammation.

Vincristine (Vin) is a well-known antitumor agent that frequently evokes neuropathic pain and decreases the quality of life of patients. Polysaccharides (GBP) extracted from Gastrodia elata Blume have been demonstrated to possess anti-inflammatory and neuroprotective effects in vivo; however, the effects of GBP on Vin-induced neuropathic pain remain unknown. The present study is aimed at exploring the alleviative potential of GBP against chemotherapy-evoked peripheral neuropathy to better understand and extend its pharmacological application. Vin was administered intraperitoneally to evoke neuropathic pain. GBP was orally administered for 21 days. The mechanical allodynia and thermal hyperalgesia were assessed using the Von Frey test and hot-plate test. Histopathological changes were assessed by hematoxylin and eosin staining. ELISA kits were used to measure the levels of inflammatory cytokines in the sciatic nerve, spinal cord, and dorsal root ganglion (DRG). qRT-PCR was employed to examine the expression of inflammatory cytokines and Sirtuin1 (SIRT1) in the sciatic nerve, spinal cord, and DRG. Our findings revealed that GBP treatment enhanced the paw withdrawal latency and paw withdrawal threshold and restored Vin-induced sciatic nerve damage in rats. GBP also attenuated the Vin-induced increase of proinflammatory cytokine levels, including IL-6, IL-8, TNF-α, IL-1ß, and NF-κB. On the molecular level, treatment with GBP downregulated the mRNA levels of IL-6, IL-8, TNF-α, and IL-1ß in the sciatic nerve, spinal cord, and DRG. Meanwhile, GBP increased SIRT1 activity and mRNA expression levels. Our data indicated that GBP exerted a potential protective effect against chemotherapy-induced neuropathic pain which might be mediated via the inhibition of neuroinflammation.

Transcriptome Reveals Roles of Lignin-Modifying Enzymes and Abscisic Acid in the Symbiosis of Mycena and Gastrodia elata.

Gastrodia elata is a well-known medicinal and heterotrophic orchid. Its germination, limited by the impermeability of seed coat lignin and inhibition by abscisic acid (ABA), is triggered by symbiosis with fungi such as Mycena spp. However, the molecular mechanisms of lignin degradation by Mycena and ABA biosynthesis and signaling in G. elata remain unclear. In order to gain insights into these two processes, this study analyzed the transcriptomes of these organisms during their dynamic symbiosis. Among the 25 lignin-modifying enzyme genes in Mycena, two ligninolytic class II peroxidases and two laccases were significantly upregulated, most likely enabling Mycena hyphae to break through the lignin seed coats of G. elata. Genes related to reduced virulence and loss of pathogenicity in Mycena accounted for more than half of annotated genes, presumably contributing to symbiosis. After coculture, upregulated genes outnumbered downregulated genes in G. elata seeds, suggesting slightly increased biological activity, while Mycena hyphae had fewer upregulated than downregulated genes, indicating decreased biological activity. ABA biosynthesis in G. elata was reduced by the downregulated expression of 9-cis-epoxycarotenoid dioxygenase (NCED-2), and ABA signaling was blocked by the downregulated expression of a receptor protein (PYL12-like). This is the first report to describe the role of NCED-2 and PYL12-like in breaking G. elata seed dormancy by reducing the synthesis and blocking the signaling of the germination inhibitor ABA. This study provides a theoretical basis for screening germination fungi to identify effective symbionts and for reducing ABA inhibition of G. elata seed germination.

GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Gastrodia elata, a fully mycoheterotrophic orchid without photosynthetic ability, only grows symbiotically with the fungus Armillaria. The mechanism of carbon distribution in this mycoheterotrophy is unknown. We detected high sucrose concentrations in all stages of Gastrodia tubers, suggesting sucrose may be the major sugar transported between fungus and orchid. Thick symplasm-isolated wall interfaces in colonized and adjacent large cells implied involvement of sucrose importers. Two sucrose transporter (SUT)-like genes, GeSUT4 and GeSUT3, were identified that were highly expressed in young Armillaria-colonized tubers. Yeast complementation and isotope tracer experiments confirmed that GeSUT4 functioned as a high-affinity sucrose-specific proton-dependent importer. Plasma-membrane/tonoplast localization of GeSUT4-GFP fusions and high RNA expression of GeSUT4 in symbiotic and large cells indicated that GeSUT4 likely functions in active sucrose transport for intercellular allocation and intracellular homeostasis. Transgenic Arabidopsis overexpressing GeSUT4 had larger leaves but were sensitive to excess sucrose and roots were colonized with fewer mutualistic Bacillus, supporting the role of GeSUT4 in regulating sugar allocation. This is not only the first documented carbon import system in a mycoheterotrophic interaction but also highlights the evolutionary importance of sucrose transporters for regulation of carbon flow in all types of plant-microbe interactions.

De Novo Assembly and Annotation of the Juvenile Tuber Transcriptome of a Gastrodia elata Hybrid by RNA Sequencing: Detection of SSR Markers.

Gastrodia elata is a traditional Chinese herbal medicine with good therapeutic effect on various nervous and cerebrovascular diseases. In the present study, we generated 20,611,556 raw reads from the young tuber transcriptome of a G. elata hybrid (Gastrodia elata BI.f.elata × Gastrodia elata BI.f.pilifera) by using Illumina HiSeq™ 4000 sequencing platform. De novo assembly and bioinformatics analysis revealed 20,237,474 clean reads, including 2,529,684,250 bp that assembled into 34,323 unigenes with an average length of 695.19 bp. Among them, 24,698 (71.96%) unigenes were annotated by at least one of the Nr, Swiss-Prot, COG and KEGG databases. A total of 4236 (12.34%) unigenes were identified as candidate transcription factors, and 2007 (5.85%) unigenes were found to contain at least one single sequence repeat (SSR). Of these SSRs, AG/CT repeat motif was the most frequent, with a total of 498 (21.67%). This study will enhance our understanding about the molecular mechanism of physiological metabolism, growth and development of G. elata, particularly abundant SSR markers will offer plenty of alternative tools for further studies about molecular genetics, molecular breeding and association analysis.

Gastrodin Protects Cardiomyocytes from Anoxia/Reoxygenation Injury by 14-3-3η.

Gastrodin (GAS) is the major component isolated from the rhizome of the Chinese traditional medicinal herb "Tianma." Many clinical studies have found that GAS protects cardiomyocytes in cardiovascular diseases, although the effects and underlying mechanisms on cardiovascular anoxia/reoxygenation (A/R) injury remain unknown. This study is aimed at exploring the effect of gastrodin on cardiomyocytes in A/R injury. Our results suggested that the protective effect of GAS on cardiomyocytes is associated with upregulated 14-3-3η levels. Pretreatment with GAS could increase the cell viability and decrease the activities of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH). GAS could also reduce reactive oxygen species (ROS) production, inhibit mitochondrial permeability transition pore (mPTP) opening, alter the maintenance of the mitochondrial membrane potential (∆Ψm), decrease the activation of caspase-3, and finally restrain cell apoptosis. Downregulating 14-3-3η levels by transfection with siRNA14-3-3η clearly attenuated the protective effect of GAS on cardiomyocytes in A/R injury.

The Phenolic Components of Gastrodia elata improve Prognosis in Rats after Cerebral Ischemia/Reperfusion by Enhancing the Endogenous Antioxidant Mechanisms.

Pharmacological or spontaneous thrombolysis in ischemic stroke triggers an outbreak of reactive oxygen species and results in neuron death. Nrf2-mediated antioxidation in cells has been proved as a pivotal target for neuroprotection. This research reports that phenolic components of Gastrodia elata Blume (PCGE), a traditional Chinese medicine, can alleviate the pathological lesions in the penumbra and hippocampus by increasing the survival of neurons and astrocytes and improve neurofunction and cognition after reperfusion in a rat model of middle cerebral artery occlusion. LDH assay indicated that pretreatment of cells with PCGE (25 µg/ml) for 24 h significantly reduced H2O2-induced cell death in astrocytes and SH-SY5Y cells. Western blot showed that the nucleus accumulation of Nrf2 and the expression of cellular HO-1 and NQO-1, two of Nrf2 downstream proteins, were increased in both cells. BDNF, an Nrf2-dependent neurotrophic factor, was also upregulated by PCGE in astrocytes. These results illustrated that PCGE can reduce the cerebral ischemia/reperfusion injury and improve prognosis by remedying the cell damage within affected tissues. The protective effects of PCGE seem to be via activation of a Nrf2-mediated cellular defense system. Therefore, PCGE could be a therapeutic candidate for ischemic stroke and other oxidative stress associated neurological disorders.

LEA proteins from Gastrodia elata enhance tolerance to low temperature stress in Escherichia coli.

BACKGROUND: Late embryogenesis abundant (LEA) proteins were initially discovered about 35years ago as accumulating late in embryogenesis of cotton seeds. Although abundant in seeds and pollens, these proteins have been found to protect cells against desiccation, cold, high temperature, and high salinity. OBJECTIVE: Here, we present the first comprehensive survey of LEA proteins and their encoding genes in Gastrodia elata, a well-known medicinal orchid in China. Moreover, we researched on LEA family evolutionary relationships and functional characteristics. METHODS: The LEA gene family in G. elata (GeLEAs) was cloned based on RNA-Seq data. In addition, all of GeLEA genes were introduced into Escherichia coli to assess the function of GeLEAs under low temperature stress. RESULTS: Based on the phylogenetic analysis with Arabidopsis and Oryza LEA proteins, we identified 8 LEA protein encoding genes in G. elata that could be classified into 6 distinct subgroups. The motif composition of these proteins was an important feature specific to LEA groups. Compared with control, the expressions of 5 GeLEAs in E. coli exhibited enhanced cold resistance and viability, indicating that GeLEAs protein could play a protective role in cells under low temperature stress. CONCLUSION: Our results suggest that LEAs from G. elata play an important role in responses to abiotic stress.

A practical protocol for comprehensive evaluation of sulfur-fumigation of Gastrodia Rhizoma using metabolome and health risk assessment analysis.

Gastrodia Rhizoma is one of the most heavily sulfur-fumigated edible and medical herbs in the marketplace. We developed a practical protocol using an ultra-performance liquid chromatography coupled with quadrupole time-of-flight-MSE (UPLC/QTOF-MSE)-based metabolome and health risk assessment model to identify characteristic sulfur-fumigated markers, dissect chemical transformation mechanisms, and control the quality of sulfur-fumigated Gastrodia Rhizoma. Two sulfur-containing p-hydroxybenzyl products, one sulfur-containing disaccharide, one glycolipid, and two phospholipids were selected and identified as markers based on multivariate statistical analysis. In particular, the sulfur-containing markers p-hydroxybenzyl hydrogen sulfite and trace p-mercaptobenzyl hydrogen sulfate were positively correlated with the active major phenolics. Moreover, a practical index the time of the minimum content was useful for evaluating the extent of the sulfur-fumigation under different weight ratios of the sulfur to herbal materials (1:20, 1:40, and 1:80). Ultimately, the 1:40 ratio within 1h of sulfur-fumigation was considered as safe and efficient for herb quality preservation under the maximum residue limit of 750mg/kg. This study shows that the practical protocol-based discriminated markers and practical limits can be applied to quality assurance of sulfur-fumigation and non-fumigation Gastrodia Rhizoma and other edible or medical materials.

Specific targeted quantification combined with non-targeted metabolite profiling for quality evaluation of Gastrodia elata tubers from different geographical origins and cultivars.

Gastrodia elata tuber (GET) has been widely used as a famous herbal medicine in China and other East Asian countries. In this work, we developed a comprehensive strategy integrating targeted and non-targeted analyses for quality evaluation and discrimination of GET from different geographical origins and cultivars. Firstly, 43 batches of GET samples of five cultivars from three regions in China were efficiently quantified by a "single standard to determine multi-components" (SSDMC) method. Six marker compounds were simultaneously determined within 11min using gastrodin as the internal standard. It showed that samples from different regions and cultivars could not be differentiated by the contents of six marker compounds. Secondly, a non-targeted metabolite profiling analysis was performed by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS). Samples from different geographical origins and cultivars were clearly discriminated by principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA). 147 discriminant ions contributing to the group separation were selected from 1194 aligned variables. Furthermore, based on the relative intensities of discriminant ions, support vector machines (SVM) was employed to predict the geographical origins of GET. The obtained SVM model showed excellent prediction performance with an average prediction accuracy of 100%. These results demonstrated that the UHPLC-QTOF/MS-based non-targeted metabolite profiling analysis, as a vital supplement to targeted analysis, can be used to discriminate the geographical origins and cultivars of GET.

Evaluation of four different analytical tools to determine the regional origin of Gastrodia elata and Rehmannia glutinosa on the basis of metabolomics study.

Chemical profiles of medicinal plants could be dissimilar depending on the cultivation environments, which may influence their therapeutic efficacy. Accordingly, the regional origin of the medicinal plants should be authenticated for correct evaluation of their medicinal and market values. Metabolomics has been found very useful for discriminating the origin of many plants. Choosing the adequate analytical tool can be an essential procedure because different chemical profiles with different detection ranges will be produced according to the choice. In this study, four analytical tools, Fourier transform near­infrared spectroscopy (FT-NIR), 1H-nuclear magnetic resonance spectroscopy (1H­NMR), liquid chromatography-mass spectrometry (LC-MS), and gas chromatography-mass spectroscopy (GC-MS) were applied in parallel to the same samples of two popular medicinal plants (Gastrodia elata and Rehmannia glutinosa) cultivated either in Korea or China. The classification abilities of four discriminant models for each plant were evaluated based on the misclassification rate and Q2 obtained from principal component analysis (PCA) and orthogonal projection to latent structures-discriminant analysis (OPLS­DA), respectively. 1H-NMR and LC-MS, which were the best techniques for G. elata and R. glutinosa, respectively, were generally preferable for origin discrimination over the others. Reasoned by integrating all the results, 1H-NMR is the most prominent technique for discriminating the origins of two plants. Nonetheless, this study suggests that preliminary screening is essential to determine the most suitable analytical tool and statistical method, which will ensure the dependability of metabolomics-based discrimination.

Metabolomics approach for the discrimination of raw and steamed Gastrodia elata using liquid chromatography quadrupole time-of-flight mass spectrometry.

A liquid chromatography quadrupole time-of-flight mass spectrometry-based metabolomics approach was applied to metabolite profiling of Gastrodia elata in order to identify raw and steamed G. elata and explore potential biomarkers for each processing state. A statistical classification method, significant analysis of microarrays, was used to select influential metabolites from the different forms. Through metabolite selection, several potential biomarkers were determined and assigned by matching mass information with that of reference compounds or by comparing it with data in the literature. Furthermore, the developed method was cross-checked using two validation procedures. The first validation was performed simultaneously with the metabolite profiling of G. elata using all detected metabolites, and the second was performed after the metabolite profiling using representative standard compounds of G. elata. Overall, this study can be applied to quality assurance of G. elata.

Gastrodin production from p-2-hydroxybenzyl alcohol through biotransformation by cultured cells of Aspergillus foetidus and Penicillium cyclopium.

The objective of this work was to take advantage of the resting cells of suitable fungus as an in vitro model to prepare gastrodin from p-2-hydroxybenzyl alcohol (HBA), which mainly exists in the metabolites of the plant Gastrodia elata Blume. The one-step biotransformation of HBA into gastrodin was examined with the filamentous fungi cells of Aspergillus foetidus and Penicillium cyclopium AS 3.4513 in this study. The fundamental conditions of biotransformation were screened and compared for both fungi. P. cyclopium AS 3.4513 had better gastrodin-producing capability than A. foetidus through one-step bioconversion. The highest yield of gastrodin was 36 mg/L for A. foetidus ZU-G1 and 65 mg/L for P. cyclopium AS 3.4513 under the respective development condition during 6 days of biotransformation. The comparative results show that P. cyclopium AS 3.4513 reveals great potential to form gastrodin using HBA as the precursor. The products catalyzed by the resting cells of P. cyclopium AS 3.4513 were identified through NMR and ESI-MS. Current results can be applied not only to the chemical synthesis processes that may involve the hydroxylation reaction but also to the industrial production. The selected fungus is the potential biocatalyst for HBA glucosylation.

Effects of acidic polysaccharides from gastrodia rhizome on systolic blood pressure and serum lipid concentrations in spontaneously hypertensive rats fed a high-fat diet.

The effects of acidic polysaccharides purified from Gastrodia rhizome on blood pressure and serum lipid levels in spontaneously hypertensive rats (SHR) fed a high-fat diet were investigated. Acidic polysaccharides were purified from crude polysaccharides by DEAE-Sepharose CL-6B. Thirty-six male SHR were randomly divided into three groups: Gastrodia rhizome crude polysaccharide (A), acidic polysaccharide (B) groups, and a control group (C). A 5-week oral administration of all treatment groups was performed daily in 3- to 8-week-old SHRs with a dose of 6 mg/kg of body weight/day. After 5 weeks of treatment, total cholesterol in the acidic polysaccharide group, at 69.7 ± 10.6 mg/dL, was lower than in the crude polysaccharide group (75.0 ± 6.0 mg/dL) and the control group (89.2 ± 7.4 mg/dL). In addition, triglyceride and low-density lipoprotein cholesterol levels in the acidic polysaccharide group were lower than in the crude polysaccharide and control groups. The atherogenic index of the acidic polysaccharide group was 46.3% lower than in the control group. Initial blood pressure after the initial three weeks on the high-fat diet averaged 195.9 ± 3.3 mmHg among all rats. Compared with the initial blood pressure, the final blood pressure in the control group was increased by 22.8 mmHg, whereas it decreased in the acidic polysaccharide group by 14.9 mmHg. These results indicate that acidic polysaccharides from Gastrodia rhizome reduce hypertension and improve serum lipid levels.

Identification of major metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside by LC/MS/MS.

N(6) -(4-hydroxybenzyl) adenine riboside, a novel neuroprotective compound found in Gastrodia elata at trace level, is regarded as a potential drug for the treatment of neural degenerative disease. To understand the metabolism of this compound, the metabolites in rat urine and plasma of N(6) -(4-hydroxybenzyl) adenine riboside were analyzed by HPLC-ESI-MS/MS after oral administration of this compound. Beside the parent compound, six phase I metabolites and four phase II metabolites in urine were detected by scanning all possible metabolites in extracted ion chromatograms mode. By comparing their product ion spectra and retention times with those of parent compound, these metabolites were identified and proved to be mainly formed via hydrolysis or hydroxylation in phase I, N-sulfation or N-glucuronidation in phase II or their combinations. Similarly, the parent compound, one phase I metabolite and two phase II metabolites were also identified in rat plasma. Therefore, the in vivo metabolic pathways of N(6) -(4-hydroxybenzyl) adenine riboside in rat were proposed.

Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena.

We investigated the physiological ecology of the Asian non-photosynthetic orchid Gastrodia confusa. We revealed its mycorrhizal partners by using molecular identification and identified its ultimate nutritional source by analysing carbon and nitrogen natural stable isotope abundances. Molecular identification using internal transcribed spacer and large subunit nrDNA sequences showed that G. confusa associates with several species of litter- and wood-decomposer Mycena fungi. The carbon and nitrogen isotope signatures of G. confusa were analysed together with photosynthetic plant reference samples and samples of the ectomycorrhizal epiparasite Monotropa uniflora. We found that G. confusa was highly enriched in (13)C but not greatly in (15)N, while M. uniflora was highly enriched in both (13)C and (15)N. The (13)C and (15)N signatures of G. confusa were the closest to those of the fruit bodies of saprotrophic fungi. Our results demonstrate for the first time using molecular and mass-spectrometric approaches that myco-heterotrophic plants gain carbon through parasitism of wood or litter decaying fungi. Furthermore, we demonstrate that, several otherwise free-living non-mycorrhizal, Mycena can be mycorrhizal partners of orchids.