Gene-Metabolite Network Analysis Revealed Tissue-Specific Accumulation of Therapeutic Metabolites in Mallotus japonicus.

Mallotus japonicus is a valuable traditional medicinal plant in East Asia for applications as a gastrointestinal drug. However, the molecular components involved in the biosynthesis of bioactive metabolites have not yet been explored, primarily due to a lack of omics resources. In this study, we established metabolome and transcriptome resources for M. japonicus to capture the diverse metabolite constituents and active transcripts involved in its biosynthesis and regulation. A combination of untargeted metabolite profiling with data-dependent metabolite fragmentation and metabolite annotation through manual curation and feature-based molecular networking established an overall metabospace of M. japonicus represented by 2129 metabolite features. M. japonicus de novo transcriptome assembly showed 96.9% transcriptome completeness, representing 226,250 active transcripts across seven tissues. We identified specialized metabolites biosynthesis in a tissue-specific manner, with a strong correlation between transcripts expression and metabolite accumulations in M. japonicus. The correlation- and network-based integration of metabolome and transcriptome datasets identified candidate genes involved in the biosynthesis of key specialized metabolites of M. japonicus. We further used phylogenetic analysis to identify 13 C-glycosyltransferases and 11 methyltransferases coding candidate genes involved in the biosynthesis of medicinally important bergenin. This study provides comprehensive, high-quality multi-omics resources to further investigate biological properties of specialized metabolites biosynthesis in M. japonicus.

Chromosome-level genome assembly of Ophiorrhiza pumila reveals the evolution of camptothecin biosynthesis.

Plant genomes remain highly fragmented and are often characterized by hundreds to thousands of assembly gaps. Here, we report chromosome-level reference and phased genome assembly of Ophiorrhiza pumila, a camptothecin-producing medicinal plant, through an ordered multi-scaffolding and experimental validation approach. With 21 assembly gaps and a contig N50 of 18.49 Mb, Ophiorrhiza genome is one of the most complete plant genomes assembled to date. We also report 273 nitrogen-containing metabolites, including diverse monoterpene indole alkaloids (MIAs). A comparative genomics approach identifies strictosidine biogenesis as the origin of MIA evolution. The emergence of strictosidine biosynthesis-catalyzing enzymes precede downstream enzymes' evolution post γ whole-genome triplication, which occurred approximately 110 Mya in O. pumila, and before the whole-genome duplication in Camptotheca acuminata identified here. Combining comparative genome analysis, multi-omics analysis, and metabolic gene-cluster analysis, we propose a working model for MIA evolution, and a pangenome for MIA biosynthesis, which will help in establishing a sustainable supply of camptothecin.

Multiomics-based characterization of specialized metabolites biosynthesis in Cornus Officinalis.

Cornus officinalis, an important traditional medicinal plant, is used as major constituents of tonics, analgesics, and diuretics. While several studies have focused on its characteristic bioactive compounds, little is known on their biosynthesis. In this study, we performed LC-QTOF-MS-based metabolome and RNA-seq-based transcriptome profiling for seven tissues of C. officinalis. Untargeted metabolome analysis assigned chemical identities to 1,215 metabolites and showed tissue-specific accumulation for specialized metabolites with medicinal properties. De novo transcriptome assembly established for C. officinalis showed 96% of transcriptome completeness. Co-expression analysis identified candidate genes involved in the biosynthesis of iridoids, triterpenoids, and gallotannins, the major group of bioactive metabolites identified in C. officinalis. Integrative omics analysis identified 45 cytochrome P450s genes correlated with iridoids accumulation in C. officinalis. Network-based integration of genes assigned to iridoids biosynthesis pathways with these candidate CYPs further identified seven promising CYPs associated with iridoids' metabolism. This study provides a valuable resource for further investigation of specialized metabolites' biosynthesis in C. officinalis.

Metabolomics with 15N Labeling for Characterizing Missing Monoterpene Indole Alkaloids in Plants.

Monoterpene indole alkaloids (MIAs) in medicinal plants remain uncharacterized owing to their complicated structure by metabolomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) despite their pharmaceutical importance. We demonstrate an untargeted metabolome analysis with 15nitrogen (N) labeling to characterize MIAs having an indolic skeleton in the flowers, leaves, petioles, stems, and roots of Catharanthus roseus. Principal component analysis using 15N- and nonlabeled metabolome data showed that N-containing metabolites (N-metabolites) are labeled with 15N. Paring of the 15N- and nonlabeled precursor ions were performed using the criteria of retention time, difference of m/z value, and a nonlabeled product ion at m/z 144.08 that indicates an indolic skeleton. The mass shift of the m/z value of the product and precursor ions to their 15N-labeled ions identified the number of N of their ions. Finally, molecular formula of 45 MIAs was unambiguously identified using the identified N number. The alkaloid network analysis using the MS/MS similarity showed the structural commonness and uniqueness among the MIAs. Of them, antirhine was identified using an authentic standard compound. Multimetabolomics using LC-MS/MS and imaging mass spectrometry showed that antirhine accumulates considerably in the epidermis and vascular cylinder of the roots. The developed approach showed the existence of the missing MIAs. The modification of this approach will identify other MIAs that contain a hydroxylated or methoxylated indolic skeleton.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

UGT79B31 is responsible for the final modification step of pollen-specific flavonoid biosynthesis in Petunia hybrida.

MAIN CONCLUSION: UGT79B31 encodes flavonol 3- O -glycoside: 2″- O -glucosyltransferase, an enzyme responsible for the terminal modification of pollen-specific flavonols in Petunia hybrida. Flavonoids are known to be involved in pollen fertility in petunia (P. hybrida) and maize (Zea mays). As a first step toward elucidating the role of flavonoids in pollen, we have identified a glycosyltransferase that is responsible for the terminal modification of petunia pollen-specific flavonoids. An in silico search of the petunia transcriptome database revealed four candidate UDP-glycosyltransferase (UGT) genes. UGT79B31 was selected for further analyses based on a correlation between the accumulation pattern of flavonol glycosides in various tissues and organs and the expression profiles of the candidate genes. Arabidopsis ugt79b6 mutants that lacked kaempferol/quercetin 3-O-glucosyl(1 â†’ 2)glucosides, were complemented by transformation with UGT79B31 cDNA under the control of Arabidopsis UGT79B6 promoter, showing that UGT79B31 functions as a flavonol 3-O-glucoside: 2″-O-glucosyltransferase in planta. Recombinant UGT79B31 protein can convert kaempferol 3-O-galactoside/glucoside to kaempferol 3-O-glucosyl(1 â†’ 2)galactoside/glucoside. UGT79B31 prefers flavonol 3-O-galactosides to the 3-O-glucosides and rarely accepted the 3-O-diglycosides as sugar acceptors. UDP-glucose was the preferred sugar donor for UGT79B31. These results indicated that UGT79B31 encodes a flavonoid 3-O-glycoside: 2″-O-glucosyltransferase. Transient expression of UGT79B31 fused to green fluorescent protein (GFP) in Nicotiana benthamiana showed that UGT79B31 protein was localized in the cytosol.

Increased oxidative stress is related to disease severity in the ALS motor cortex: A PET study.

OBJECTIVE: To investigate cerebral oxidative stress based on an over-reductive state caused by mitochondrial dysfunction and its relationship to disease severity in patients with amyotrophic lateral sclerosis (ALS) using PET with [(62)Cu]diacetyl-bis(N(4)-methylthiosemicarbazone) ((62)Cu-ATSM). METHODS: Twelve patients with ALS and 9 age-matched healthy controls underwent a 20-minute dynamic brain PET scan after (62)Cu-ATSM injection. The standardized uptake value (SUV) images obtained from the last 10 minutes of frames were normalized by the global mean (nSUV). Regional (62)Cu-ATSM retention in the nSUV images was compared between groups using statistical parametric mapping (SPM) and region of interest (ROI) analysis. Secondary analyses evaluated the correlations between regional nSUVs and the clinical characteristics of the participants. RESULTS: In SPM mapping, patients with ALS showed a significantly greater accumulation of (62)Cu-ATSM compared to controls in the bilateral cortices around the central sulcus, including the motor cortex, and the right superior parietal lobule. ROI analysis also revealed significantly greater nSUVs in patients than controls in these regions. Increases in nSUV for these regions were associated with decreases in the revised ALS Functional Rating Scale score, suggesting a good correlation with the severity of ALS. In controls, age was correlated with nSUV for the bilateral cortices around the central sulcus, although this correlation was not observed in patients with ALS. CONCLUSIONS: (62)Cu-ATSM PET imaging demonstrated increased oxidative stress based on an over-reductive state, primarily in the motor cortex, in patients with ALS. The magnitude of oxidative stress correlated well with clinical severity, indicating that it may be associated with neurodegenerative changes in ALS.

Potential of the FES-hERL PET reporter gene system -- basic evaluation for gene therapy monitoring.

PURPOSE: In vivo reporter genes can be powerful tools in supporting and ensuring the success of gene therapy. A careful and rational design of a reporter system is essential to realize a noninvasive in vivo reporter gene imaging system applicable for humans. We designed a new in vivo reporter gene imaging system that uses F-18-labeled estradiol (FES) and human estrogen receptor ligand (hERL) binding domain, taking advantage that FES is a radiopharmaceutical already being used for human studies with access to a wide range of tissues, including the brain, and that hERL lacking DNA binding domain can no longer work as a transcription factor, and carried out basic studies to evaluate its potential for gene therapy monitoring. METHODS: We constructed a plasmid (pTIER) to coexpress a model therapeutic gene and the reporter gene hERL and transfected Cos7 cells and examined their uptake of [(3)H]estradiol and FES in culture media. The uptake of FES by mouse calf muscle electroporated with pTIER was also tested. RESULTS: The cells transfected with pTIER took up the radioligands efficiently and specifically in culture media. Also, the mouse calf muscle electroporated with pTIER accumulated a higher amount of FES than did the control. CONCLUSION: The data indicate that our new reporter gene system seems promising for in vivo imaging of gene expression and gene therapy monitoring.

Gender differences in cerebral glucose metabolism: a PET study.

OBJECTIVE: Some studies have examined gender differences in brain function based on cerebral blood flow and cerebral metabolism by using positron emission tomography (PET). However, the findings of these studies are controversial and most of them were analyzed by the regions of interest (ROIs) method. Here, we evaluated gender differences of cerebral glucose metabolism under the resting state in a voxel-based analysis. METHODS: We studied 44 healthy volunteers (22 females, 63.0+/-6.3 years, and 22 males, 63.1+/-8.4 years). Cerebral glucose metabolic images were obtained with (18)F-fluorodeoxyglucose (FDG) and PET. All individual data were transformed to standard brain space and the male and female groups were compared using statistical parametric mapping (SPM). RESULTS: The males had significantly higher glucose metabolism in the right insula, middle temporal gyrus, and medial frontal lobe than the females. Glucose metabolism in the hypothalamus was significantly higher in females than in males. There was a significant correlation between aging and glucose metabolism in the left thalamus in males and in the left caudate nucleus and hypothalamus in females. In males, but not females, there was a significant asymmetry between the bilateral hemispheres. CONCLUSION: We found that there were obvious gender differences in regional cerebral glucose metabolism and this is the first report of higher glucose metabolism in the hypothalamus in females than in males.