The Integration of the Metabolome and Transcriptome for Dendrobium nobile Lindl. in Response to Methyl Jasmonate.

Dendrobium nobile Lindl., as an endangered medicinal plant within the genus Dendrobium, is widely distributed in southwestern China and has important ecological and economic value. There are a variety of metabolites with pharmacological activity in D. nobile. The alkaloids and polysaccharides contained within D. nobile are very important active components, which mainly have antiviral, anti-tumor, and immunity improvement effects. However, the changes in the compounds and functional genes of D. nobile induced by methyl jasmonate (MeJA) are not clearly understood. In this study, the metabolome and transcriptome of D. nobile were analyzed after exposure to MeJA. A total of 377 differential metabolites were obtained through data analysis, of which 15 were related to polysaccharide pathways and 35 were related to terpenoids and alkaloids pathways. Additionally, the transcriptome sequencing results identified 3256 differentially expressed genes that were discovered in 11 groups. Compared with the control group, 1346 unigenes were differentially expressed in the samples treated with MeJA for 14 days (TF14). Moreover, the expression levels of differentially expressed genes were also significant at different growth and development stages. According to GO and KEGG annotations, 189 and 99 candidate genes were identified as being involved in terpenoid biosynthesis and polysaccharide biosynthesis, respectively. In addition, the co-expression analysis indicated that 238 and 313 transcription factors (TFs) may contribute to the regulation of terpenoid and polysaccharide biosynthesis, respectively. Through a heat map analysis, fourteen terpenoid synthetase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were identified that may be related to dendrobine biosynthesis. Among them, one sesquiterpene synthase gene was found to be highly expressed after the treatment with MeJA and was positively correlated with the content of dendrobine. This study provides important and valuable metabolomics and transcriptomic information for the further understanding of D. nobile at the metabolic and molecular levels and provides candidate genes and possible intermediate compounds for the dendrobine biosynthesis pathway, which lays a certain foundation for further research on and application of Dendrobium.

Comparison of the Metabolomics of Different Dendrobium Species by UPLC-QTOF-MS.

Dendrobium Sw. (family Orchidaceae) is a renowned edible and medicinal plant in China. Although widely cultivated and used, less research has been conducted on differential Dendrobium species. In this study, stems from seven distinct Dendrobium species were subjected to UPLC-QTOF-MS/MS analysis. A total of 242 metabolites were annotated, and multivariate statistical analysis was employed to explore the variance in the extracted metabolites across the various groups. The analysis demonstrated that D. nobile displays conspicuous differences from other species of Dendrobium. Specifically, D. nobile stands out from the remaining six taxa of Dendrobium based on 170 distinct metabolites, mainly terpene and flavonoid components, associated with cysteine and methionine metabolism, flavonoid biosynthesis, and galactose metabolism. It is believed that the variations between D. nobile and other Dendrobium species are mainly attributed to three metabolite synthesis pathways. By comparing the chemical composition of seven species of Dendrobium, this study identified the qualitative components of each species. D. nobile was found to differ significantly from other species, with higher levels of terpenoids, flavonoids, and other compounds that are for the cardiovascular field. By comparing the chemical composition of seven species of Dendrobium, these qualitative components have relevance for establishing quality standards for Dendrobium.

Genome-wide identification, characterization and transcriptional profile of the SWEET gene family in Dendrobium officinale.

BACKGROUND: Dendrobium officinale Kimura et Migo (D. officinale) is a well-known traditional Chinese medicine with high content polysaccharides in stems. The SWEET (Sugars Will Eventually be Exported Transporters) family is a novel class of sugar transporters mediating sugar translocation among adjacent cells of plants. The expression patterns of SWEETs and whether they are associated with stress response in D. officinale remains uncovered. RESULTS: Here, 25 SWEET genes were screened out from D. officinale genome, most of which typically contained seven transmembrane domains (TMs) and harbored two conserved MtN3/saliva domains. Using multi-omics data and bioinformatic approaches, the evolutionary relationship, conserved motifs, chromosomal location, expression patterns, correlationship and interaction network were further analyzed. DoSWEETs were intensively located in nine chromosomes. Phylogenetic analysis revealed that DoSWEETs were divided into four clades, and conserved motif 3 specifically existed in DoSWEETs from clade II. Different tissue-specific expression patterns of DoSWEETs suggested the division of their roles in sugar transport. In particular, DoSWEET5b, 5c, and 7d displayed relatively high expression levels in stems. DoSWEET2b and 16 were significantly regulated under cold, drought, and MeJA treatment, which were further verified using RT-qPCR. Correlation analysis and interaction network prediction discovered the internal relationship of DoSWEET family. CONCLUSIONS: Taken together, the identification and analysis of the 25 DoSWEETs in this study provide basic information for further functional verification in D. officinale.

Uncovering the involvement of DoDELLA1-interacting proteins in development by characterizing the DoDELLA gene family in Dendrobium officinale.

BACKGROUND: Gibberellins (GAs) are widely involved in plant growth and development. DELLA proteins are key regulators of plant development and a negative regulatory factor of GA. Dendrobium officinale is a valuable traditional Chinese medicine, but little is known about D. officinale DELLA proteins. Assessing the function of D. officinale DELLA proteins would provide an understanding of their roles in this orchid's development. RESULTS: In this study, the D. officinale DELLA gene family was identified. The function of DoDELLA1 was analyzed in detail. qRT-PCR analysis showed that the expression levels of all DoDELLA genes were significantly up-regulated in multiple shoots and GA3-treated leaves. DoDELLA1 and DoDELLA3 were significantly up-regulated in response to salt stress but were significantly down-regulated under drought stress. DoDELLA1 was localized in the nucleus. A strong interaction was observed between DoDELLA1 and DoMYB39 or DoMYB308, but a weak interaction with DoWAT1. CONCLUSIONS: In D. officinale, a developmental regulatory network involves a close link between DELLA and other key proteins in this orchid's life cycle. DELLA plays a crucial role in D. officinale development.

Preparation of Dendrobium officinale Flower Anthocyanin and Extended Lifespan in Caenorhabditis elegans.

The Dendrobium officinale flower is a non-medicinal part of the plant, rich in a variety of nutrients and bioactive ingredients. The purpose of this article was to explore the preparation conditions of anthocyanins (ACNs) from the D. officinale flower. Subsequently, its anti-aging effects were evaluated with Caenorhabditis elegans. Results showed that the ACNs had antioxidant activities on scavenging free radicals (DPPH· and ABTS+·), and the clearance rate was positively correlated with the dose. Additionally, ACNs significantly increased the activity of superoxide dismutase (SOD) in C. elegans, which was 2.068-fold higher than that of the control. Treatment with ACNs at 150 µL extended the lifespan of C. elegans by 56.25%, and treatment with ACNs at 50 µL promoted fecundity in C. elegans. Finally, the protective effect of ACNs enhanced stress resistance, thereby increasing the survival numbers of C. elegans, which provided insights for the development and practical application of functional products.

Evolution patterns of NBS genes in the genus Dendrobium and NBS-LRR gene expression in D. officinale by salicylic acid treatment.

BACKGROUND: Dendrobium officinale Kimura et Migo, which contains rich polysaccharides, flavonoids and alkaloids, is a Traditional Chinese Medicine (TCM) with important economic benefits, while various pathogens have brought huge losses to its industrialization. NBS gene family is the largest class of plant disease resistance (R) genes, proteins of which are widely distributed in the upstream and downstream of the plant immune systems and are responsible for receiving infection signals and regulating gene expression respectively. It is of great significance for the subsequent disease resistance breeding of D. officinale to identify NBS genes by using the newly published high-quality chromosome-level D. officinale genome. RESULTS: In this study, a total of 655 NBS genes were uncovered from the genomes of D. officinale, D. nobile, D. chrysotoxum, V. planifolia, A. shenzhenica, P. equestris and A. thaliana. The phylogenetic results of CNL-type protein sequences showed that orchid NBS-LRR genes have significantly degenerated on branches a and b. The Dendrobium NBS gene homology analysis showed that the Dendrobium NBS genes have two obvious characteristics: type changing and NB-ARC domain degeneration. Because the NBS-LRR genes have both NB-ARC and LRR domains, 22 D. officinale NBS-LRR genes were used for subsequent analyses, such as gene structures, conserved motifs, cis-elements and functional annotation analyses. All these results suggested that D. officinale NBS-LRR genes take part in the ETI system, plant hormone signal transduction pathway and Ras signaling pathway. Finally, there were 1,677 DEGs identified from the salicylic acid (SA) treatment transcriptome data of D. officinale. Among them, six NBS-LRR genes (Dof013264, Dof020566, Dof019188, Dof019191, Dof020138 and Dof020707) were significantly up-regulated. However, only Dof020138 was closely related to other pathways from the results of WGCNA, such as pathogen identification pathways, MAPK signaling pathways, plant hormone signal transduction pathways, biosynthetic pathways and energy metabolism pathways. CONCLUSION: Our results revealed that the NBS gene degenerations are common in the genus Dendrobium, which is the main reason for the diversity of NBS genes, and the NBS-LRR genes generally take part in D. officinale ETI system and signal transduction pathways. In addition, the D. officinale NBS-LRR gene Dof020138, which may have an important breeding value, is indirectly activated by SA in the ETI system.

Light and Potassium Improve the Quality of Dendrobium officinale through Optimizing Transcriptomic and Metabolomic Alteration.

BACKGROUND: Dendrobium officinale is a perennial epiphytic herb in Orchidaceae. Cultivated products are the main alternative for clinical application due to the shortage of wild resources. However, the phenotype and quality of D. officinale have changed post-artificial cultivation, and environmental cues such as light, temperature, water, and nutrition supply are the major influencing factors. This study aims to unveil the mechanisms beneath the cultivation-induced variation by analyzing the changes of the metabolome and transcriptome of D. officinale seedlings treated with red- blue LED light and potassium fertilizer. RESULTS: After light- and K-treatment, the D. officinale pseudobulbs turned purple and the anthocyanin content increased significantly. Through wide-target metabolome analysis, compared with pseudobulbs in the control group (P), the proportion of flavonoids in differentially-accumulated metabolites (DAMs) was 22.4% and 33.5% post light- and K-treatment, respectively. The gene modules coupled to flavonoids were obtained through the coexpression analysis of the light- and K-treated D. officinale transcriptome by WGCNA. The KEGG enrichment results of the key modules showed that the DEGs of the D. officinale pseudobulb were enriched in phenylpropane biosynthesis, flavonoid biosynthesis, and jasmonic acid (JA) synthesis post-light- and K-treatment. In addition, anthocyanin accumulation was the main contribution to the purple color of pseudobulbs, and the plant hormone JA induced the accumulation of anthocyanins in D. officinale. CONCLUSIONS: These results suggested that light and potassium affected the accumulation of active compounds in D. officinale, and the gene-flavone network analysis emphasizes the key functional genes and regulatory factors for quality improvement in the cultivation of this medicinal plant.

Genome-wide identification and characterization of active ingredients related ß-Glucosidases in Dendrobium catenatum.

BACKGROUND: Dendrobium catenatum/D. officinale (here after D. catenatum), a well-known economically important traditional medicinal herb, produces a variety of bioactive metabolites including polysaccharides, alkaloids, and flavonoids with excellent pharmacological and clinical values. Although many genes associated with the biosynthesis of medicinal components have been cloned and characterized, the biosynthetic pathway, especially the downstream and regulatory pathway of major medicinal components in the herb, is far from clear. ß-glucosidases (BGLUs) comprise a diverse group of enzymes that widely exist in plants and play essential functions in cell wall modification, defense response, phytohormone signaling, secondary metabolism, herbivore resistance, and scent release by hydrolyzing ß-D-glycosidic bond from a carbohydrate moiety. The recent release of the chromosome-level reference genome of D. catenatum enables the characterization of gene families. Although the genome-wide analysis of the BGLU gene family has been successfully conducted in various plants, no systematic analysis is available for the D. catenatum. We previously isolated DcBGLU2 in the BGLU family as a key regulator for polysaccharide biosynthesis in D. catenatum. Yet, the exact number of DcBGLUs in the D. catenatum genome and their possible roles in bioactive compound production deserve more attention. RESULTS: To investigate the role of BGLUs in active metabolites production, 22 BGLUs (DcBGLU1-22) of the glycoside hydrolase family 1 (GH1) were identified from D. catenatum genome. Protein prediction showed that most of the DcBGLUs were acidic and phylogenetic analysis classified the family into four distinct clusters. The sequence alignments revealed several conserved motifs among the DcBGLU proteins and analyses of the putative signal peptides and N-glycosylation site revealed that the majority of DcBGLU members dually targeted to the vacuole and/or chloroplast. Organ-specific expression profiles and specific responses to MeJA and MF23 were also determined. Furthermore, four DcBGLUs were selected to test their involvement in metabolism regulation. Overexpression of DcBGLU2, 6, 8, and 13 significantly increased contents of flavonoid, reducing-polysaccharide, alkaloid and soluble-polysaccharide, respectively. CONCLUSION: The genome-wide systematic analysis identified candidate DcBGLU genes with possible roles in medicinal metabolites production and laid a theoretical foundation for further functional characterization and molecular breeding of D. catenatum.

Differential expression of ethylene biosynthetic and receptor genes in pollination-induced senescence of Dendrobium flowers.

Following successful pollination, Dendrobium orchid flowers rapidly undergo senescence. In Dendrobium cv. Khao Chaimongkol, compatible pollination resulted in faster ethylene production and more rapid development of senescence symptoms, such as drooping, epinasty, venation and yellowing, compared with non-pollinated controls or pollination with incompatible pollinia. The DenACS1 and DenACO1 genes in the perianth of florets that had been pollinated with compatible pollinia were expressed more highly than those in non-pollinated open florets. Incompatible pollinia reduced the expression of DenACS1 and DenACO1 genes in the perianth. Transcript levels of the ethylene receptor gene DenERS1 and signaling genes DenEIL1 and DenERF1 showed differential spatial regulation with greater expression in the perianth than in the column plus ovary following compatible pollination. Compatible pollinia increased ethylene production concomitant with premature senescence and the increased expression of the DenACS1 and DenACO1 genes, and suppressed the ethylene receptor gene DenERS1, whereas incompatible pollinia did not stimulate ethylene production nor induce premature senescence but induced higher expression of DenERS1 both in the perianth and in the column plus ovary. These results suggest that the increased ethylene production in open florets pollinated with compatible pollen was partially due to an increase in the expression of DenACS1 and DenACO1 genes. The compatible pollinia induced a negative regulation of DenERS1 which may play an important role in ethylene perception and in modulating ethylene signaling transduction during pollinia-induced flower senescence.

Secondary Metabolites in the Dendrobium heterocarpum Methanolic Extract and Their Impacts on Viability and Lipid Storage of 3T3-L1 Pre-Adipocytes.

Although many natural products have proven their potential to regulate obesity through the modulation of adipocyte biology, none of them has yet been approved for clinical use in obesity therapy. This work aims to isolate valuable secondary metabolites from an orchid species (Dendrobium heterocarpum) and evaluate their possible roles in the growth and differentiation of 3T3-L1 pre-adipocytes. Six compounds were isolated from the orchid's methanolic extracts and identified as amoenylin (1), methyl 3-(4-hydroxyphenyl) propionate (2), 3,4-dihydroxy-5,4'-dimethoxybibenzyl (3), dendrocandin B (4), dendrofalconerol A (5), and syringaresinol (6). Among these phytochemicals, compounds 2, 3, and 6 exhibited lower effects on the viability of 3T3-L1 cells, offering non-cytotoxic concentrations of ≲10 µM. Compared to others tested, compound 3 was responsible for the maximum reduction of lipid storage in 3T3-L1 adipocytes (IC50 = 6.30 ± 0.10 µM). A set of protein expression studies unveiled that compound 3 at non-cytotoxic doses could suppress the expression of some key transcription factors in adipocyte differentiation (i.e., PPARγ and C/EBPα). Furthermore, this compound could deactivate some proteins involved in the MAPK pathways (i.e., JNK, ERK, and p38). Our findings prove that D. heterocarpum is a promising source to explore bioactive molecules capable of modulating adipocytic growth and development, which can potentially be assessed and innovated further as pharmaceutical products to defeat obesity.

Alkaloids from Dendrobium and their biosynthetic pathway, biological activity and total synthesis.

BACKGROUND: Dendrobium Sw. has been used for thousands of years in China as a precious traditional Chinese medicine. It is derived from stems of various Dendrobium plants and has the functions of nourishing Yin and clearing heat, activating water and nourishing the stomach, moistening the lung and relieving cough. Modern phytochemical studies show that the main components of Dendrobium include alkaloids, polysaccharides, terpenoids, diphenylbenzene, and phenanthrene. Alkaloids are natural products with obvious biological activity and are important effective components of the medicinal activity or toxicity of plants. At present, dozens of alkaloids with various structures have been isolated from Dendrobium plants, and the alkaloid contents in Dendrobium plants of different species are quite different. From the perspective of food safety, the type, molecular structure, content and potential physiological activity or toxicity of alkaloids are important bases for evaluating the safety of edible plants. Studies have shown that the alkaloids isolated from Dendrobium have neuroprotective, anti-inflammatory and antitumor activities, showing that these alkaloids with potential medicinal activity are important sources of lead compounds in innovative drug development. PURPOSE: To summarize the research progress on alkaloids in Dendrobium and provide a reference for research on the food safety and medicinal development of Dendrobium. METHOD: Information about alkaloids from Dendrobium was collected from the scientific databases Web of Science, PubChem and PubMed. We discuss the biosynthetic pathway, biological activities and total synthesis of alkaloids from Dendrobium from 1964 to 2020 and summarize the knowledge of alkaloids from Dendrobium, the biosynthetic pathway, biological activities and total synthesis. We chose publications on their chemistry, drug effects, pharmacology, metabolism and biosynthesis, physiology and toxicity. Alkaloids, Dendrobium, biosynthetic pathway and biological activities were used as keywords to extract the relevant literature. CONCLUSION: In this paper, the structural classification, biological activity, target and toxicology and synthesis of the alkaloids in Dendrobium were systematically reviewed, which will provide a reference for the safety, development and application of Dendrobium.

Dendrobium nobile Lindley Administration Attenuates Atopic Dermatitis-like Lesions by Modulating Immune Cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that can significantly affect daily life by causing sleep disturbance due to extreme itching. In addition, if the symptoms of AD are severe, it can cause mental disorders such as ADHD and suicidal ideation. Corticosteroid preparations used for general treatment have good effects, but their use is limited due to side effects. Therefore, it is essential to minimize the side effects and study effective treatment methods. Dendrobium nobile Lindley (DNL) has been widely used for various diseases, but to the best of our knowledge, its effect on AD has not yet been proven. In this study, the inhibitory effect of DNL on AD was confirmed in a DNCB-induced Balb/c mouse. In addition, the inhibitory efficacy of inflammatory cytokines in TNF-α/IFN-γ-induced HaCaT cells and PMACI-induced HMC-1 cells was confirmed. The results demonstrated that DNL decreased IgE, IL-6, IL-4, scratching behavior, SCORAD index, infiltration of mast cells and eosinophils and decreased the thickness of the skin. Additionally, DNL inhibited the expression of cytokines and inhibited the MAPK and NF-κB signaling pathways. This suggests that DNL inhibits cytokine expression, protein signaling pathway, and immune cells, thereby improving AD symptoms in mice.

Flavonoid biosynthesis in four Dendrobium species based on transcriptome sequencing and metabolite analysis.

BACKGROUND: Dendrobium is a genus of plants used as traditional Chinese herbal medicines, with high economic and medicinal value. METHODS AND RESULTS: To reveal the mechanism of flavonoid biosynthesis in Dendrobium, the metabolites and transcriptomes of four Dendrobium species (D. chrysotoxum, D. nobile, D. fimbriatum, and D. denneanum) were analyzed comprehensively. Ultra-high-performance liquid chromatography-tandem mass spectrometry analysis revealed ten flavonoid compounds in Dendrobium. In total, 100,096 unigenes were obtained from the transcript database of the four Dendrobium species. Among the identified differentially expressed genes, 51 were associated with flavonoid biosynthesis, and 670 differentially expressed transcription factors were predicted, including 194 MYB, 87 bHLH, and 100 WRKY family transcription factors, respectively. Transcriptome analysis showed that the expression levels of structural genes such as chalcone synthase (CHS), cinnamate-4-hydroxylase (C4H), and flavonoid 3'-hydroxylase (F3'H) were lower in D. chrysotoxum, D. nobile, and D. fimbriatum than those in D. denneanum, which may be the main reason for the low flavonoid contents in D. chrysotoxum, D. nobile, and D. fimbriatum. CONCLUSIONS: The expression level of structural genes corresponded to the accumulation level of flavonols in the different Dendrobium species. The results deepen the understanding of the molecular mechanism of flavonoid biosynthesis in Dendrobium and provide novel insights into the synthesis and accumulation of flavonoids in Dendrobium.

Dendrobium catenatum Lindl. Water Extracts Attenuate Atherosclerosis.

OBJECTIVES: Dendrobium catenatum Lindl. (DH) is a Chinese herbal medicine, which is often used to make tea to improve immunity in China. Rumor has it that DH has a protective effect against cardiovascular disease. However, it is not clear how DH can prevent cardiovascular disease, such as atherosclerosis (AS). Therefore, the purpose of this study is to study whether DH can prevent AS and the underlying mechanisms. METHODS: Zebrafish larvae were fed with high-cholesterol diet (HCD) to establish a zebrafish AS model. Then, we used DH water extracts (DHWE) to pretreat AS zebrafish. The plaque formation was detected by HE, EVG, and oil red O staining. Neutrophil and macrophage counts were calculated to evaluate the inflammation level. Reactive oxygen species (ROS) activity, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity in zebrafish were measured to reflect oxidative stress. The cholesterol accumulation and the levels of lipid, triglyceride (TG), and total cholesterol (TC) were measured to reflect lipid metabolism disorder. Then, parallel flow chamber was utilized to establish a low shear stress- (LSS-) induced endothelial cell (EC) dysfunction model. EA.hy926 cells were exposed to LSS (3 dyn/cm2) for 30 min and treated with DHWE. The levels of ROS, SOD, MDA, glutathione (GSH), and glutathiol (GSSG) in EA.hy926 cells were analysed to determine oxidative stress. The release of nitric oxide (NO), endothelin-1 (ET-1), and epoprostenol (PGI2) in EA.hy926 cells was measured to reflect EC dysfunction. The mRNA expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in EA.hy926 cells was detected to reflect EC dysfunction inflammation. RESULTS: The results showed that DHWE significantly reduced cholesterol accumulation and macrophage infiltration in early AS. Finally, DHWE significantly alleviate the lipid metabolism disorder, oxidative stress, and inflammation to reduce the plaque formation of AS zebrafish larval model. Meanwhile, we also found that DHWE significantly improved LSS-induced EC dysfunction and oxidative stress in vitro. CONCLUSION: Our results indicate that DHWE could be used as a prevention method to prevent AS.

JA signal-mediated immunity of Dendrobium catenatum to necrotrophic Southern Blight pathogen.

BACKGROUND: Dendrobium catenatum belongs to the Orchidaceae, and is a precious Chinese herbal medicine. In the past 20 years, D. catenatum industry has developed from an endangered medicinal plant to multi-billion dollar grade industry. The necrotrophic pathogen Sclerotium delphinii has a devastating effection on over 500 plant species, especially resulting in widespread infection and severe yield loss in the process of large-scale cultivation of D. catenatum. It has been widely reported that Jasmonate (JA) is involved in plant immunity to pathogens, but the mechanisms of JA-induced plant resistance to S. delphinii are unclear. RESULTS: In the present study, the role of JA in enhancing D. catenatum resistance to S. delphinii was investigated. We identified 2 COI1, 13 JAZ, and 12 MYC proteins in D. catenatum genome. Subsequently, systematic analyses containing phylogenetic relationship, gene structure, protein domain, and motif architecture of core JA pathway proteins were conducted in D. catenatum and the newly characterized homologs from its closely related orchid species Phalaenopsis equestris and Apostasia shenzhenica, along with the well-investigated homologs from Arabidopsis thaliana and Oryza sativa. Public RNA-seq data were investigated to analyze the expression patterns of D. catenatum core JA pathway genes in various tissues and organs. Transcriptome analysis of MeJA and S. delphinii treatment showed exogenous MeJA changed most of the expression of the above genes, and several key members, including DcJAZ1/2/5 and DcMYC2b, are involved in enhancing defense ability to S. delphinii in D. catenatum. CONCLUSIONS: The findings indicate exogenous MeJA treatment affects the expression level of DcJAZ1/2/5 and DcMYC2b, thereby enhancing D. catenatum resistance to S. delphinii. This research would be helpful for future functional identification of core JA pathway genes involved in breeding for disease resistance in D. catenatum.

Dendrobium mixture attenuates renal damage in rats with diabetic nephropathy by inhibiting the PI3K/Akt/mTOR pathway.

Dendrobium mixture (DMix) is a Traditional Chinese Medicine widely used for preventing and treating diabetic nephropathy (DN). Autophagy contributes to DN development and progression. The present study aimed to investigate the mechanism underlying the protective effects of DMix on the kidneys of rats with DN and to determine whether this involves autophagy. Herein, a high­sugar and high­fat diet, combined with the intra­abdominal injection of low­dose streptozocin, was used to induce DN in 40 Sprague­Dawley male rats. In total, 10 additional rats were used as controls. The rats with DN were then randomly divided into three groups and treated with DMix, gliquidone or saline via gastric administration for 8 weeks. Body weight, kidney weight, kidney index, fasting blood glucose (FBG), blood lipid, hemoglobin A1c (HbA1c), insulin, blood urea nitrogen and serum creatinine levels, as well as the 24­h urinary albumin excretion rate (UAER) were measured. H&E, Periodic Acid­Schiff and Masson staining were used to examine the renal pathology. The mRNA and protein expression levels of LC3 and Beclin­1 in renal tissues were measured using reverse transcription­quantitative PCR and immunohistochemistry, respectively. Western blotting was conducted to measure the protein expression levels of PI3K, phosphorylated (p)­PI3K, Akt, p­Akt, mTOR, p­mTOR, LC3 and Beclin­1 in renal tissues. It was found that DMix significantly reduced the FBG, blood lipids, HbA1c and insulin levels, kidney weight, kidney index and UAER in rats with DN, as well as improved renal function. Rats with DN showed notable glomerular hypertrophy, an increase in mesangial matrix content and renal interstitial fibrosis. Moreover, DMix notably reduced kidney damage. The results demonstrated that DMix inhibited the phosphorylation of PI3K, Akt and mTOR in the kidney tissues of rats with DN, and increased the protein and mRNA expression levels of LC3 and Beclin­1. Therefore, it was suggested that DMix has protective effects on the kidney of rats with DN, which may be associated with the inhibition of the PI3K/Akt/mTOR signaling pathway and activation of renal autophagy by this traditional medicine.

Transcriptome and Metabolome Reveal Salt-Stress Responses of Leaf Tissues from Dendrobium officinale.

Dendrobium officinale Kimura et Migo is a precious traditional Chinese medicine. Despite D. officinale displaying a good salt-tolerance level, the yield and growth of D. officinale were impaired drastically by the increasing soil secondary salinization. The molecular mechanisms of D. officinale plants' adaptation to salt stress are not well documented. Therefore, in the present study, D. officinale plants were treated with 250 mM NaCl. Transcriptome analysis showed that salt stress significantly altered various metabolic pathways, including phenylalanine metabolism, flavonoid biosynthesis, and α-linolenic acid metabolism, and significantly upregulated the mRNA expression levels of DoAOC, DoAOS, DoLOX2S, DoMFP, and DoOPR involved in the jasmonic acid (JA) biosynthesis pathway, as well as rutin synthesis genes involved in the flavonoid synthesis pathway. In addition, metabolomics analysis showed that salt stress induced the accumulation of some compounds in D. officinale leaves, especially flavonoids, sugars, and alkaloids, which may play an important role in salt-stress responses of leaf tissues from D. officinale. Moreover, salt stress could trigger JA biosynthesis, and JA may act as a signal molecule that promotes flavonoid biosynthesis in D. officinale leaves. To sum up, D. officinale plants adapted to salt stress by enhancing the biosynthesis of secondary metabolites.

The methyl jasmonate-responsive transcription factor DobHLH4 promotes DoTPS10, which is involved in linalool biosynthesis in Dendrobium officinale during floral development.

Linalool is an aromatic monoterpene produced in the Chinese medicinal plant Dendrobium officinale, but little information is available on the regulation of linalool biosynthesis. Here, a novel basic helix-loop-helix (bHLH) transcription factor, DobHLH4 from D. officinale, was identified and functionally characterized. The expression profile of DobHLH4 was positively correlated with that of DoTPS10 (R2 = 0.985, p < 0.01), which encodes linalool synthase that is responsible for linalool production, during floral development. DobHLH4 was highly expressed in petals, and was significantly induced by methyl jasmonate. Analysis of subcellular localization showed that DobHLH4 was located in the nucleus. Yeast one-hybrid and dual-luciferase assays indicated that DobHLH4 bound directly to the DoTPS10 promoter harboring the G-box element, and up-regulated DoTPS10 expression. A yeast two-hybrid screen confirmed that DobHLH4 physically interacted with DoJAZ1, suggesting that DobHLH4 might function in the jasmonic acid-mediated accumulation of linalool. Furthermore, transient overexpression of DobHLH4 in D. officinale petals significantly increased linalool production by triggering linalool biosynthetic pathway genes, especially DoTPS10. We suggest a hypothetical model that depicts how jasmonic acid signaling may regulate DoTPS10 by interacting with DobHLH4 and DoJAZ1. In doing so, the formation of linalool is controlled. Our results indicate that DobHLH4 is a positive regulator of linalool biosynthesis and may be a promising target for in vitro-based metabolic engineering to produce linalool.

Dendrobium huoshanense stem polysaccharide ameliorates rheumatoid arthritis in mice via inhibition of inflammatory signaling pathways.

The present study explored the beneficial effect of Dendrobium huoshanense stem polysaccharide (cDHPS) after oral administration on rheumatoid arthritis (RA) using type Ⅱ collagen-induced arthritis (CIA) mouse model. It was found that cDHPS effectively alleviated joint swelling, synovial hyperplasia, pannus formation, cartilage erosion and bone destruction in CIA mice. Concurrently, cDHPS remodeled the balance of Th17 and regulatory T cells, reduced the secretion of pro-inflammatory mediators related to fibroblast-like synoviocyte activation, angiogenesis, articular cartilage degradation and osteoclast differentiation, inhibited HIF-1α expression and promoted anti-inflammatory mediator release in the joint tissues and serum of CIA mice. Western blot of joint tissues showed that cDHPS significantly inhibited the phosphorylation of IκB, p65, JNK, p38, ERK1/2, AKT, PI3K, JAK1 and STAT3 in CIA mice. These results suggest that cDHPS possesses the potential of ameliorating RA and its anti-RA effect may be attributed to the inhibition of inflammatory signaling pathways.

Rapid propagation in vitro and accumulation of active substances of endangered Dendrobium cariniferum Rchb. f.

Dendrobium cariniferum is a valuable ornamental and medicinal plant rich with polysaccharides, alkaloid, and other bioactive compounds, which are potential raw materials for pharmacological utilization. In this study, an efficient protocol for the rapid propagation of D. cariniferum was developed. By using the tissue culture protocol, the effects of pH, hormone combinations, temperatures, light intensity, culture time protocorm proliferation, seedlings rooting, and accumulation of biomass with bioactive compounds were investigated. The experiments showed that the medium [1/2 MS + activated carbon1.0 g/L+ agar strip 7.5 g/L + sucrose 25 g/L] effectively promoted the germination of D. cariniferum seeds. The optimal culture conditions were found at pH 5.7, temperature 23 ± 2°C, and light intensity of 1000 Lx in the protocorm proliferation stage. Adding 1.5 g/L peptone in the medium effectively promoted the seedling rooting. The optimal culture conditions for accumulation of bioactive compounds (polysaccharides and alkaloids) of seedlings were found at temperature of 25 ± 2°C, light intensity of 1500-2000 Lx after the 60-day (d). Our study constructed a rapid propagation system in vitro for D. cariniferum, as well as the methods for efficient accumulation of active substances in seedling culture, which will serve as guidance for industrial production of D. cariniferum seedlings for both medicinal raw materials and ornamental plants. In addition, our study provided a new idea that we can directly use the high bioactive compound seedlings to extract medicinal components in industry conditions without transferring to the field.