Terpenoid Backbone Research Articles

The first chromosome-level genome assembly and transcriptome sequencing provide insights into cantharidin production of the blister beetles.

Blister beetles (Coleoptera: Meloidae) produce a natural defensive toxin cantharidin (CTD), which has been used for various cancer treatments and other diseases. Currently, the lack of chromosome-level reference genomes in Meloidae limits further understanding of the mechanism of CTD biosynthesis and environmental adaptation. In this study, the chromosome-level genome assembly of Mylabris phalerata was generated based on PacBio and Hi-C sequencing. This reference genome was about 136.68 Mb in size with contig N50 of 9.17 Mb and composed of 12 chromosomes. In comparison to six other Coleoptera insects, M. phalerata exhibited multiple expanded gene families enriched in juvenile hormone (JH) biosynthetic process pathway, farnesol dehydrogenase activity, and cytochrome P450, which may be related to CTD biosynthesis. Consistently, the transcriptomic analysis suggested the "terpenoid backbone biosynthesis" pathway and "the juvenile hormone" as putative core pathways of CTD biosynthesis and presented eight up-regulated differential expression genes in male adults as candidate genes. It is possible that the restricted feeding niche and lifestyle of M. phalerata were the cause of the gene family's contraction of odorant binding proteins. The ABC transporters (ABCs) related to exporting bound toxins out of the cell and the resistance to the self-secreted toxins (e.g. CTD) were also contracted, possibly due to other self-protection strategies in M. phalerata. A foundation of understanding CTD biosynthesis and environmental adaptation of blister beetles will be established by our reference genome and discoveries.

HS-SPEM-GC-MS Analyses of Volatiles and Transcriptome Analysis of Camellia tetracocca in Response to Feeding by Empoasca onukii Matsuda

The tea leafhopper (Empoasca (Matsumurasca) onukii Matsuda, 1952) is the predominant pest infesting tea and causes significant losses in yield. Plants have evolved multiple defense responses to protect themselves from insect herbivores. Camellia tetracocca, a kind of ancient tea with local popularity, is unique to southwest China. This ancient tea is often attacked by the tea leafhopper. However, there are no relevant reports on changes in volatile components and defense signaling pathways after Camellia tetracocca has been attacked by E. onukii. To reveal changes in the responses of the volatile components of C. tetracocca to E. onukii, the headspace sampling technique and GC-MS were adopted to determine and analyze the volatiles. Furthermore, to unveil the defense mechanisms of C. tetracocca in response to E. onukii, transcriptome analysis was applied to damaged tea leaves and healthy leaves. The GC-MS analysis showed significant increases in the linalool and (E)-2-hexenal contents. The transcriptome analysis revealed a set of core pest-responsive genes (3074 upregulated and 5098 downregulated). Most of the enriched pest-responsive genes are mainly involved in plant hormone signal transduction photosynthesis, terpenoid backbone biosynthesis, alpha-linolenic acid metabolism, and the NF-κB signaling pathway. A conjoint analysis of the transcriptome and metabolome indicated that N2-acetylornithine, D-malic acid, theogallin, fucosyllactose, 5-caffeoylquinic acid, fumarprotocetraric acid, and arabinogalactose have a significant correlation with LOC114279735, LOC114317534, LOC114282078, LOC114317768, and LOC114263181. This study lays a foundation for further research on the breeding and cultivation of insect-resistant germplasm resources for C. tetracocca and on the promotion and development of the plant.

Meta-analysis of transcriptome reveals key genes relating to oil quality in olive

BackgroundOlive oil contains monounsaturated oleic acid up to 83% and phenolic compounds, making it an excellent source of fat. Due to its economic importance, the quantity and quality of olive oil should be improved in parallel with international standards. In this study, we analyzed the raw RNA-seq data with a meta-analysis approach to identify important genes and their metabolic pathways involved in olive oil quality.ResultsA deep search of RNA-seq published data shed light on thirty-nine experiments associated with the olive transcriptome, four of these proved to be ideal for meta-analysis. Meta-analysis confirmed the genes identified in previous studies and released new genes, which were not identified before. According to the IDR index, the meta-analysis had good power to identify new differentially expressed genes. The key genes were investigated in the metabolic pathways and were grouped into four classes based on the biosynthetic cycle of fatty acids and factors that affect oil quality. Galactose metabolism, glycolysis pathway, pyruvate metabolism, fatty acid biosynthesis, glycerolipid metabolism, and terpenoid backbone biosynthesis were the main pathways in olive oil quality. In galactose metabolism, raffinose is a suitable source of carbon along with other available sources for carbon in fruit development. The results showed that the biosynthesis of acetyl-CoA in glycolysis and pyruvate metabolism is a stable pathway to begin the biosynthesis of fatty acids. Key genes in oleic acid production as an indicator of oil quality and critical genes that played an important role in production of triacylglycerols were identified in different developmental stages. In the minor compound, the terpenoid backbone biosynthesis was investigated and important enzymes were identified as an interconnected network that produces important precursors for the synthesis of a monoterpene, diterpene, triterpene, tetraterpene, and sesquiterpene biosynthesis.ConclusionsThe results of the current investigation can produce functional data related to the quality of olive oil and would be a useful step in reducing the time of cultivar screening by developing gene specific markers in olive breeding programs, releasing also new genes that could be applied in the genome editing approach.

Targeted metabolome and transcriptome analyses reveal changes in gibberellin and related cell wall-acting enzyme-encoding genes during stipe elongation in Flammulina filiformis.

Flammulina filiformis, a typical agaric fungus, is a widely cultivated and consumed edible mushroom. Elongation of its stipe (as the main edible part) is closely related to its yield and commercial traits; however, the endogenous hormones during stipe elongation and their regulatory mechanisms are not well understood. Gibberellin (GA) plays an important role in the regulation of plant growth, but little has been reported in macro fungi. In this study, we first treated F. filiformis stipes in the young stage with PBZ (an inhibitor of GA) and found that PBZ significantly inhibited elongation of the stipe. Then, we performed GA-targeted metabolome and transcriptome analyses of the stipe at both the young and elongation stages. A total of 13 types of GAs were detected in F. filiformis; the contents of ten of them, namely, GA3, GA4, GA8, GA14, GA19, GA20, GA24, GA34, GA44, and GA53, were significantly decreased, and the contents of three (GA5, GA9, and GA29) were significantly increased during stipe elongation. Transcriptome analysis showed that the genes in the terpenoid backbone biosynthesis pathway showed varying expression patterns: HMGS, HMGR, GPS, and FPPS were significantly upregulated, while CPS/KS had no significant difference in transcript level during stipe elongation. In total, 37 P450 genes were annotated to be involved in GA biosynthesis; eight of them were upregulated, twelve were downregulated, and the rest were not differentially expressed. In addition, four types of differentially expressed genes involved in stipe elongation were identified, including six signal transduction genes, five cell cycle-controlling genes, twelve cell wall-related enzymes and six transcription factors. The results identified the types and content of GAs and the expression patterns of their synthesis pathways during elongation in F. filiformis and revealed the molecular mechanisms by which GAs may affect the synthesis of cell wall components and the cell cycle of the stipe through the downstream action of cell wall-related enzymes, transcription factors, signal transduction and cell cycle control, thus regulating stipe elongation. This study is helpful for understanding the roles of GAs in stipe development in mushrooms and lays the foundation for the rational regulation of stipe length in agaric mushrooms during production.

Integrated 16S rRNA sequencing and nontargeted metabolomics analysis to reveal the mechanisms of Yu-Ye Tang on type 2 diabetes mellitus rats.

Yu-Ye Tang (YYT) is a classical formula widely used in treatment of type 2 diabetes mellitus (T2DM). However, the specific mechanism of YYT in treating T2DM is not clear. The aim of this study was to investigate the therapeutic effect of YYT on T2DM by establishing a rat model of T2DM. The mechanism of action of YYT was also explored through investigating gut microbiota and serum metabolites. The results indicated YYT had significant therapeutic effects on T2DM. Moreover, YYT could increase the abundance of Lactobacillus, Candidatus_Saccharimonas, UCG-005, Bacteroides and Blautia while decrease the abundance of and Allobaculum and Desulfovibrio in gut microbiota of T2DM rats. Nontargeted metabolomics analysis showed YYT treatment could regulate arachidonic acid metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, glycerophospholipid metabolism, pentose and glucuronate interconversions, phenylalanine metabolism, steroid hormone biosynthesis, terpenoid backbone biosynthesis, tryptophan metabolism, and tyrosine metabolism in T2DM rats. In conclusion, our research showed that YYT has a wide range of therapeutic effects on T2DM rats, including antioxidative and anti-inflammatory effects. Furthermore, YYT corrected the altered gut microbiota and serum metabolites in T2DM rats. This study suggests that YYT may have a therapeutic impact on T2DM by regulating gut microbiota and modulating tryptophan and glycerophospholipid metabolism, which are potential key pathways in treating T2DM.

Transcriptome analysis provides insights into coumarin biosynthesis in the medicinal plant Angelica dahurica cv. Yubaizhi

Angelica dahurica roots have a long history of use in traditional Chinese medicine due to their high coumarin content. To address the increasing demand for these roots, a synthetic biology approach has been proposed. Nevertheless, our comprehension of coumarin biosynthesis and its regulation remains limited. In this study, we utilized Hiseq2500 sequencing to analyze the transcriptomes of A. dahurica at different growth stages while concurrently quantifying coumarin content. Differentially expressed gene (DEG) analysis was employed to identify key genes involved in coumarin and terpenoid backbone biosynthesis. Weighted gene co-expression network analysis (WGCNA) was applied to identify gene modules strongly associated with coumarin content, elucidating the regulatory relationships between transcription factors (TFs) and pathway genes. Furthermore, KEGG enrichment analysis was used to explore essential pathways governing coumarin biosynthesis, with the identification of hub genes. Our results indicated that total coumarin content was highest in the roots, followed by leaves and stems, across all three developmental stages. Transcriptome analysis identified a total of 92,478 genes, among which 215 and 30 genes were implicated in coumarin and terpenoid backbone biosynthesis, respectively. Within the 73 identified gene modules by WGCNA, three modules—namely aquamarine1 (comprising two OMTs, one CSE, one AACT, one HDS, two PSs, one 2OGO, four UGTs, and seven CYP450s), darkmagenta (containing one UGT and one HDR), and navajowhite2 (consisting of one HCT, three UGTs, one CYP71A25, one OMT, one CSE, one HDS, and one PT)—were strongly associated with imperatorin, oxypeucedanin, and isoimperatorin content, respectively. KEGG enrichment analysis highlighted significant enrichment of cytochrome P450, transporter, and ubiquitin system pathways. Moreover, TF-gene regulatory analysis unveiled the complexity of coumarin biosynthesis, with 17 TF families regulating 17 genes in the aquamarine1 module, 8 TF families regulating 2 genes in the darkmagenta module, and 8 TF families regulating 7 genes in the navajowhite2 module. These comprehensive findings provide valuable insights into coumarin biosynthesis in A. dahurica, facilitating future research and potential applications in traditional Chinese medicine and synthetic biology strategies.

Analysis of Pharmacological Activities and Mechanisms of Essential Oil in Leaves of C. grandis 'Tomentosa' by GC-MS/MS and Network Pharmacology.

Citrus grandis 'Tomentosa,' a fruit epicarp of C. grandis 'Tomentosa' or C. grandis (L.) Osbeck is widely used in health food and medicine. Based on our survey results, there are also rich essential oils with bioactivities in leaves, but the chemical compounds in this part and relevant pharmacological activities have never been studied systematically. Therefore, this study was to preliminarily decipher the pharmacological activities and mechanisms of the essential oil in leaves of C. grandis 'Tomentosa' by an integrated network pharmacology approach. Essential oil compositions from leaves ofC. grandis 'Tomentosa' were identified using GC-MS/MS. And then, the targets of these oil compositions were predicted and screened from TCMSP, SwissTargetPrediction, STITCH and SEA databases. STRING database was used to construct the protein-protein interaction networks, and the eligible protein targets were input into WebGestalt 2019 to carry out GO enrichment and KEGG pathway enrichment analysis. Based on the potential targets, disease enrichment information was obtained by TTD databases. Cytoscape software was used to construct the component-target-disease network diagrams. Finally, 61 essential oil chemical components were identified by GC-MS/MS, which correspond to 679 potential targets. Biological function analysis showed 12, 19, and 12 GO entries related to biological processes, cell components and molecular functions, respectively. 43 KEGG pathways were identified, of which the most significant categories were terpenoid backbone biosynthesis, TNF signaling pathway and leishmaniasis. The component-target-disease network diagram revealed that the essential oil compositions in leaves of C. grandis 'Tomentosa' could treat tumors, immune diseases, neurodegenerative diseases and respiratory diseases, which were highly related to CHRM1, PTGS2, CASP3, MAP2K1 and CDC25B. This study may provide new insight into C. grandis 'Tomentosa' or C. grandis (L.) Osbeck and may provide useful information for future utilization and development.

Integrated transcriptomic and metabolomic analysis reveals the molecular basis of tissue-specific accumulation of bioactive steroidal alkaloids in Fritillaria unibracteata

Fritillaria unibracteata is an endangered medicinal plant whose bulb has been used as a Chinese herb to suppress cough, asthma and excessive phlegm for centuries. Steroidal alkaloids, which are synthesized via the steroid synthesis pathways, are their significant bioactive constituents. However, few studies on genes involved in steroidal alkaloid biosynthesis in F. unibracteata have been reported, mainly due to the lack of the F. unibracteata genome. In this paper, comparative transcriptomic and metabolomic analyses of four different tissues of F. unibracteata (leaves, flowers, stems, and bulbs) were performed. Imperialine, peiminine, and peimisine were among the significant bioactive compounds that were considerably abundant in bulb tissue, according to the metabolomic findings. Then, 83.60 Gb transcriptome sequencing of four different tissues was performed, of which one gene encoding phosphomevalonate kinase was directly functionally characterized to verify the accuracy of sequences obtained from the transcriptome. A total of 9217 differentially expressed unigenes (DEGs) were identified in four different tissues of F. unibracteata. GO and KEGG enrichments revealed that phenylpropanoid biosynthesis, MVA-mediated terpenoid backbone biosynthesis, and steroid biosynthesis were enriched in bulb tissue, whereas enrichment of MEP-mediated terpenoid backbone biosynthesis, photosynthesis, photosynthesis-antenna protein and carotenoid biosynthesis was observed in aerial tissues. Moreover, clustering analysis indicated that the downstream steroid biosynthesis pathway was more important in steroidal alkaloid biosynthesis compared to the upstream terpenoid backbone biosynthesis pathway. Hence, MVA-mediated biosynthesis of steroidal alkaloids was proposed, in which 15 bulb-clustered DEGs were positively correlated with a high accumulation of bioactive steroid alkaloids, further validating our proposal. In addition, 36 CYP450s showing a positive correlation with bioactive steroidal alkaloids provided candidate enzymes to catalyze the subsequent steps of steroidal alkaloid biosynthesis. In addition, the transcription factors and ABC transporters clustered in bulb tissue might be responsible for the regulation and transportation of steroidal alkaloid biosynthesis. Protein-protein interaction analysis implied a highly complex steroid alkaloid biosynthesis network in which delta (24)-sterol reductase might be one of the central catalysts. Based on the integrated transcriptome and metabolome, this current study is a first step in understanding the tissue-specific biosynthesis of steroidal alkaloids in F. unibracteata. Furthermore, key genes and regulators identified herein could facilitate metabolic engineering to improve steroidal alkaloids in F. unibracteata.

Investigation of sex expression profiles and the cantharidin biosynthesis genes in two blister beetles.

Cantharidin (CTD) is a well-established defensive toxin synthesized by blister beetles, displaying both therapeutic potential and toxicity. Among these beetles, Hycleus cichorii and Hycleus phaleratus are the two most commercially significant species due to their capacity to produce CTD in males. In this investigation, we conducted a gene expression profiling analysis of male and female individuals of these two species, utilizing the Illumina Hiseq4000 platform. We identified 7,983 expressed genes, including 2,823 differentially expressed genes (DEGs) shared by both male and female blister beetles. Nineteen genes related to CTD biosynthesis in the terpenoid backbone biosynthesis pathway were identified, including hydroxymethylglutaryl-CoA reductase (HMGR; EC:1.1.1.34), which demonstrated a significant correlation with CTD content. Furthermore, hydroxymethylglutaryl-CoA synthase (HMGS; EC:2.3.3.10) and isopentenyl-diphosphate Delta-isomerase (IDI; EC:5.3.3.2) were also found to be significantly up-regulated in males. Comparative analysis revealed that NADP+-dependent farnesol dehydrogenase (FOHSDR; EC:1.1.1.216) and farnesyl diphosphate synthase (FDPS; EC:2.5.1.1) had the highest copy number in these beetles, significantly higher than the copy number of the other four non-Meloidae insects. The analysis of the protein-protein interaction network of genes related to CTD biosynthesis revealed that the acetyl-CoA C-acetyltransferase (ACAT; EC:2.3.1.9) gene was the central gene, exhibiting greater expression in male blister beetles than in females. This study offers novel insights into the mechanisms of CTD biosynthesis in blister beetles and enhances our comprehensions of the association between particular genes and CTD content.

Integrative transcriptome and metabolome analysis reveals the mechanisms of light-induced pigmentation in purple waxy maize.

Waxy maize, mainly consumed at the immature stage, is a staple and vegetable food in Asia. The pigmentation in the kernel of purple waxy maize enhances its nutritional and market values. Light, a critical environmental factor, affects anthocyanin biosynthesis and results in pigmentation in different parts of plants, including in the kernel. SWL502 is a light-sensitive waxy maize inbred line with purple kernel color, but the regulatory mechanism of pigmentation in the kernel resulting in purple color is still unknown. In this study, cyanidin, peonidin, and pelargonidin were identified as the main anthocyanin components in SWL502, evaluated by the ultra-performance liquid chromatography (UPLC) method. Investigation of pigment accumulation in the kernel of SWL502 was performed at 12, 17, and 22 days after pollination (DAP) under both dark and light treatment conditions via transcriptome and metabolome analyses. Dark treatment affected genes and metabolites associated with metabolic pathways of amino acid, carbohydrate, lipid, and galactose, biosynthesis of phenylpropanoid and terpenoid backbone, and ABC transporters. The expression of anthocyanin biosynthesis genes, such as 4CL2, CHS, F3H, and UGT, was reduced under dark treatment. Dynamic changes were identified in genes and metabolites by time-series analysis. The genes and metabolites involved in photosynthesis and purine metabolism were altered in light treatment, and the expression of genes and metabolites associated with carotenoid biosynthesis, sphingolipid metabolism, MAPK signaling pathway, and plant hormone signal transduction pathway were induced by dark treatment. Light treatment increased the expression level of major transcription factors such as LRL1, myc7, bHLH125, PIF1, BH093, PIL5, MYBS1, and BH074 in purple waxy maize kernels, while dark treatment greatly promoted the expression level of transcription factors RVE6, MYB4, MY1R1, and MYB145. This study is the first report to investigate the effects of light on waxy maize kernel pigmentation and the underlying mechanism at both transcriptome and metabolome levels, and the results from this study are valuable for future research to better understand the effects of light on the regulation of plant growth.

Identification of potential hub genes associated with atopic dermatitis-like recombinant human epidermal model using integrated transcriptomic and proteomic analysis.

Atopic dermatitis (AD) is a severe inflammatory skin disorder, characterized by elevated levels of proinflammatory cytokines that fuel a vicious cycle of inflammation. While inflammatory recombinant human epidermal (RHE) models relevant to AD have been established, comprehensive understanding remains limited. To illuminate changes and identify potential hub genes involved in AD-related inflammation, RHE models, stimulated by an inflammatory cocktail including polyinosinic-polycytidylic acid, tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4) and interleukin 13 (IL-13), were constructed and examined using tandem mass tags-proteomic coupled with RNA-seq transcriptomic analyses. Principal component analysis (PCA), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway functional enrichment were employed for the analysis of related genes and proteins. Protein-protein interaction networks helped identify hub genes, which were further confirmed by qPCR and western blot. We observed high expression of thymic stromal lymphopoietin in the inflammatory RHE. Our study identified 2369 differentially expressed genes and 880 differentially expressed proteins in the cocktail-induced group versus the normal control group. A total of 248 overlapping symbols were enriched in various biological processes and signaling pathways, including cornification envelope, cell-cell junction, calcium ion binding, extracellular matrix receptor, terpenoid backbone biosynthesis, and peroxisome proliferator-activated receptors signaling pathway, among others. Among the 248 overlapping symbols, CytoHubba identified 10 hub molecules, namely signal transducer and activator of transcription 3 (STAT3), integrin subunit beta 1 (ITGB1), filaggrin (FLG), involucrin (IVL), DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 (DDX58), small proline rich protein 1B (SPRR1B), interferon induced with helicase C domain 1 (IFIH1), desmoglein 1 (DSG1), collagen type XVII alpha 1 chain (COL17A1), and integrin subunit alpha 6 (ITGA6), based on the degree. These integrated results offer valuable insights into the molecular mechanisms of AD and present potential tools for screening cosmetic formulations intended for the treatment of AD.

Transcriptome and physiological analysis highlights the hormone, phenylpropanoid, and photosynthesis effects on early somatic embryogenesis in Ginkgo biloba

The somatic embryogenesis (SEs) system in Ginkgo biloba L. has not been fully established due to the recalcitrance of this species, and it has seriously limited the application of SEs technology, such as elite varieties large-scale production, gene editing, or transformation. Recently, we have induced the cotyledon-shaped somatic embryos (SE) of G. biloba, which was so far observed the closest to mature somatic embryos. Here, we performed the physiological analysis in six cultured stages during SEs formation, including d-NEC (non-embryogenic callus), d-EC (embryogenic callus), d-SE, d-WHC (while hairy callus), L-EC (EC in light), and L-SE (SE in light). The transcriptomic analysis in d-NEC, d-EC, L-EC, and d-WHC was also carried out. We found that the higher (IAA+ZR+iPA)/ABA and the lower IAA/(iPA+ZR) levels were critical in the somatic embryogenesis of G. biloba. The higher levels of soluble sugar, total flavonoid, terpene lactone, PAL, F3'H, and CCR were beneficial to somatic embryogenesis. In addition, the peroxidase (POD) might be related to the cell wall formation of somatic embryos in G. biloba. A total of 36517 unigenes were identified in four groups by transcriptome analysis, and genes differentially expressed (DEGs) were related to hormone homeostasis i.e., SAUR50, Jasmonic acid synthesis i.e., OPR3, phenylpropanoid biosynthesis i.e., CCR1, POD43 and C4H, terpenoid backbone biosynthesis i.e., HMGS, starch and sucrose metabolism, i.e., coniferin β-glucosidase, and photosynthesis, i.e., D1 protein. Furthermore, the majority of identified up-regulated transcription factors (TFs) such as MYB4, WRKY43, EFR8, and WUS might be responsible for the acquisition of SEs. The light was helpful to the early formation of somatic embryos, and the gene GbWUS1 and GbCLV3 were detected with higher expression in early SE induction of G. biloba. This finding will provide important theoretical and technical support for gene editing, cell totipotency research, and rapid propagation in G. biloba.

Transcriptome-Based Analysis of the Liver Response Mechanism of Black Porgy (Acanthopagrus schlegelii) to Stocking Density

Long-term high stocking density often brings negative effects such as decreased body weight, decreased immunity, and increased mortality to cultured fish, while the effects of short-term stocking are relatively less studied. In this experiment, we characterized spatial and temporal gene expression in black porgy (Acanthopagrus schlegelii) liver by establishing two stocking density groups—high (H: 26.5 kg/m3) and low (L: 2.95 kg/m3)—and conducting transcriptome sequencing before collecting liver samples at two time points: 6 h (AL and AH) and 60 d (CL and CH) into the culture trial. There were 648 and 550 differentially expressed genes (DEGs) in the AL-vs-AH and CL-vs-CH groups, respectively. The DEGs in the AL-vs-AH group were significantly enriched in steroid biosynthesis, terpenoid backbone biosynthesis, the PPAR signaling pathway, proteasome, aminoacyl-tRNA biosynthesis, and ribosome biogenesis in eukaryotes. The DEGs in the CL-vs-CH group were more significantly enriched in lipid metabolism-related pathways, such as the PPAR signaling pathway, fatty acid metabolism, and cholesterol metabolism. In addition, immune-related pathways such as the phagosome and complement and coagulation cascades were also enriched in the CL-vs-CH group. The expression changes concerning the DEGs in each group were further analyzed. The DEGs related to steroid synthesis, protein synthesis, and the degradation pathways were down-regulated, but immune-related genes were generally up-regulated in the AH group. The DEGs related to lipid synthesis were significantly up-regulated in the CH group, but the DEGs related to lipid consumption and utilization were down-regulated. The expression of immune-related DEGs was also negatively affected in the CH group. These results suggest that acute and chronic crowding stress affects lipid metabolism and immune regulation in the liver of black porgy. Acute stress particularly affected hepatic protein metabolism, while chronic stress showed more effects on hepatic lipid metabolism. The results of this study provide a theoretical basis for understanding the effects of high stocking densities upon black porgy and may also provide a reference for crowding stress regulation studies in other fish species.

Comparative transcriptome analysis to identify putative genes involved in carvacrol biosynthesis pathway in two species of Satureja, endemic medicinal herbs of Iran.

Satureja is rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemic Satureja species of Iran, containing different yields (Satureja khuzistanica and Satureja rechingeri). Cross-species differential expression analysis was conducted between two species of Satureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified for S. khuzistanica and S. rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns of S. khuzistanica and S. rechingeri transcripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors (such as MYC4, bHLH, and ARF18) that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report on de novo assembly and transcriptome data analysis in Satureja which could be useful for an understanding of the main constituents of Satureja essential oil and future research in this genus.

Molecular Mechanism of miR160d in Regulating Kiwifruit Resistance to Botrytis cinerea.

Gray mold caused by Botrytis cinerea leads to huge economic losses to the kiwifruit (Actinidia chinensis) industry. Elucidating the molecular mechanism responding to B. cinerea is the theoretical basis for the resistance to molecular breeding of kiwifruit. Previous studies have shown that miR160 regulates plant disease resistance through the indole-3-acetic acid (IAA) signaling pathway. In this study, kiwifruit "Hongyang" was used as the material, and Ac-miR160d and its target genes were identified and cloned. Overexpression and virus-induced gene silencing (VIGS) technology combined with RNA-seq were adopted to analyze the regulatory role of Ac-miR160d in kiwifruit resistance to B. cinerea. Silencing Ac-miR160d (AcMIR160d-KN) increased kiwifruit sensitivity to B. cinerea, whereas overexpression of Ac-miR160d (AcMIR160d-OE) increased kiwifruit resistance to B. cinerea, suggesting that Ac-miR160d positively regulates kiwifruit resistance to B. cinerea. In addition, overexpression of Ac-miR160d in kiwifruit increased antioxidant enzyme activities, such as catalase (CAT) and superoxide dismutase (SOD), and endogenous phytohormone IAA and salicylic acid (SA) content, in response to B. cinerea-induced stress. RNA-seq identified 480 and 858 unique differentially expressed genes in the AcMIR160d-KN vs CK and AcMIR160d-OE vs CK groups, respectively, with fold change ≥2 and false discovery rate <0.01. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that families associated with "biosynthesis of secondary metabolites" are possibly regulated by Ac-miR160d. "Phenylpropanoid biosynthesis", "flavonoid biosynthesis", and "terpenoid backbone biosynthesis" were further activated in the two comparison groups upon B. cinerea infection. Our results may reveal the molecular mechanism by which miR160d regulates kiwifruit resistance to B. cinerea and may provide gene resources for molecular breeding in kiwifruit resistance.

Combined Analysis of Volatile Compounds and Extraction of Floral Fragrance Genes in Two Dendrobium Species

Many species of the Dendrobium genus are traditional Chinese herbal medicine and ornamental plants. Flower fragrance is one of the most important horticultural ornamental characters and plays a crucial role in the ecology, economy, and aesthetics of plants. However, the volatile constituents and key regulatory genes related to floral biosynthesis are poorly understood. In this experiment, the flowers from two species of Dendrobium with high-scent smells, Dendrobium moniliforme (L.) Sw. (D. moniliforme), and light-scent smells, Dendrobium nobile “H1” (D. “H1”), were selected. The aim of this study was to explore the key gene expression profiles of floral biosynthesis by combining volatile constituent determination and transcriptome analysis in two different Dendrobium species. Physiological determination results showed that 60 volatile compounds were identified in D. moniliforme and 52 volatile compounds were identified in D. ‘H1’ flowers in four flowering stages, and the full bloom stage was the most complicated stage because there were 41 and 33 volatile compounds, respectively. These compounds belong to terpenes, aromatics, fatty acids, nitrogenous compounds, ketones, alcohols, and alkanes, respectively. The components identified in the gynandrium and petals revealed that the petals were probably the most important sites affecting the release of volatiles. The relative content of terpene compounds was the highest, with 77.25% (D. moniliforme) and 50.38% (D. “H1”), respectively. Transcriptome analysis showed that differentially expressed genes (DEGs) were highly enriched in terpenoid backbone biosynthesis and that four linalool synthetase (LIS) genes were up-regulated in high-scent smell species. This study is helpful to explore the key genes of flower fragrance and provides a theoretical basis for further understanding of the regulatory molecular functions of floral synthesis and release, as well as for the cultivation of new aromatic species.

Changes in terpene biosynthesis and submergence tolerance in cotton

BackgroundFlooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown.ResultsThe transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress.ConclusionsRegulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.

Identification of two terpenoids that accumulate in Chinese water chestnut in response to fresh-cut processing.

As a form of vegetable in China, freshly cut corms of Chinese water chestnuts (Eleocharis dulcis) are well received by consumers. Few studies have investigated the metabolites present in fresh-cut E. dulcis, particularly during the storage stage. Two compounds, triterpenoids and apocarotenoids, were identified in fresh-cut E. dulcis during the late storage period using thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and nuclear magnetic resonance (NMR) spectroscopy. The content of these two compounds gradually increased in the surface tissue of fresh-cut E. dulcis during storage. Moreover, the transcript levels of 10 genes involved in terpenoid backbone biosynthesis and five genes involved in carotenoid precursor biosynthesis were evaluated via quantitative real-time PCR (qRT-PCR). Expression of the rate-limiting enzyme-coding genes CwDXS and CwHMGS was significantly induced by wounding. CwMYC and CwbHLH18, which belong to bHLH transcription factors (TFs) IIIe and VIa subgroup, were isolated from E. dulcis corm. Phylogenetic analysis showed that CwMYC and CwbHLH18 grouped with other terpenoid-regulated bHLHs, and their transcript levels were strongly induced after fresh-cut processing. These results suggested that the biosynthesis of terpenoids and apocarotenoids in fresh-cut E. dulcis strongly depended on the transcriptional regulation of structural genes involved in the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways. However, the complex secondary metabolism of fresh-cut E. dulcis during late storage requires further investigation.

De Novo Transcriptome Profiling for the Generation and Validation of Microsatellite Markers, Transcription Factors, and Database Development for Andrographis paniculata.

Andrographis paniculata belongs to the family Acanthaceae and is known for its medicinal properties owing to the presence of unique constituents belonging to the lactones, diterpenoids, diterpene glycosides, flavonoids, and flavonoid glycosides groups of chemicals. Andrographolide, a major therapeutic constituent of A. paniculata, is extracted primarily from the leaves of this plant and exhibits antimicrobial and anti-inflammatory activities. Using 454 GS-FLX pyrosequencing, we have generated a whole transcriptome profile of entire leaves of A. paniculata. A total of 22,402 high-quality transcripts were generated, with an average transcript length and N50 of 884 bp and 1007 bp, respectively. Functional annotation revealed that 19,264 (86%) of the total transcripts showed significant similarity with the NCBI-Nr database and were successfully annotated. Out of the 19,264 BLAST hits, 17,623 transcripts were assigned GO terms and distributed into three major functional categories: molecular function (44.62%), biological processes (29.19%), and cellular component (26.18%) based on BLAST2GO. Transcription factor analysis showed 6669 transcripts, belonging to 57 different transcription factor families. Fifteen TF genes that belong to the NAC, MYB, and bHLH TF categories were validated by RT PCR amplification. In silico analysis of gene families involved in the synthesis of biochemical compounds having medicinal values, such as cytochrome p450, protein kinases, heat shock proteins, and transporters, was completed and a total of 102 different transcripts encoding enzymes involved in the biosynthesis of terpenoids were predicted. Out of these, 33 transcripts belonged to terpenoid backbone biosynthesis. This study also identified 4254 EST-SSRs from 3661 transcripts, representing 16.34% of the total transcripts. Fifty-three novel EST-SSR markers generated from our EST dataset were used to assess the genetic diversity among eighteen A. paniculata accessions. The genetic diversity analysis revealed two distinct sub-clusters and all accessions based on the genetic similarity index were distinct from each other. A database based on EST transcripts, EST-SSR markers, and transcription factors has been developed using data generated from the present study combined with available transcriptomic resources from a public database using Meta transcriptome analysis to make genomic resources available in one place to the researchers working on this medicinal plant.

Trichome-Specific Analysis and Weighted Gene Co-Expression Correlation Network Analysis (WGCNA) Reveal Potential Regulation Mechanism of Artemisinin Biosynthesis in Artemisia annua.

Trichomes are attractive cells for terpenoid biosynthesis and accumulation in Artemisia annua. However, the molecular process underlying the trichome of A. annua is not yet fully elucidated. In this study, an analysis of multi-tissue transcriptome data was performed to examine trichome-specific expression patterns. A total of 6646 genes were screened and highly expressed in trichomes, including artemisinin biosynthetic genes such as amorpha-4,11-diene synthase (ADS) and cytochrome P450 monooxygenase (CYP71AV1). Mapman and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that trichome-specific genes were mainly enriched in lipid metabolism and terpenoid metabolism. These trichome-specific genes were analyzed by a weighted gene co-expression network analysis (WGCNA), and the blue module linked to terpenoid backbone biosynthesis was determined. Hub genes correlated with the artemisinin biosynthetic genes were selected based on TOM value. ORA, Benzoate carboxyl methyltransferase (BAMT), Lysine histidine transporter-like 8 (AATL1), Ubiquitin-like protease 1 (Ulp1) and TUBBY were revealed as key hub genes induced by methyl jasmonate (MeJA) for regulating artemisinin biosynthesis. In summary, the identified trichome-specific genes, modules, pathways and hub genes provide clues and shed light on the potential regulatory mechanisms of artemisinin biosynthesis in trichomes in A. annua.