Multi-omics analysis reveals the evolutionary origin of diterpenoid alkaloid biosynthesis pathways in Aconitum.

Diterpenoid alkaloids (DAs) have been often utilized in clinical practice due to their analgesic and anti-inflammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown. In this study, we successfully assembled a high-quality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum (A. vilmorinianum) (5.76 Gb). An A. vilmorinianum-specific whole-genome duplication event was discovered using comparative genomic analysis, which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A. vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.

Correction: Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids.

Correction for 'Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids' by Yong Shen et al., Nat. Prod. Rep., 2020, 37, 763-796, https://doi.org/10.1039/D0NP00002G.

Molecular mechanisms and evolutionary history of phytomelatonin in flowering.

Flowering is a critical stage in plant life history, which is coordinated by environmental signals and endogenous cues. Phytomelatonin is a widely distributed indoleamine present in all living organisms and plays pleiotropic roles in plant growth and development. Recent evidence has established that phytomelatonin could modulate flowering in many species, probably in a concentration-dependent manner. Phytomelatonin seems to associate with floral meristem identification and floral organ formation, and the fluctuation of phytomelatonin might be important for flowering. Regarding the underlying mechanisms, phytomelatonin interacts with the central components of floral gene regulatory networks directly or indirectly, including the MADS-box gene family, phytohormones, and reactive oxygen species (ROS). From an evolutionary point of view, the actions of phytomelatonin in flowering probably evolved during the period of the diversification of flowering plants and could be regarded as a functional extension of its primary activities. The presumed evolutionary history of phytomelatonin-modulated flowering is proposed, presented in the chronological order of the appearance of phytomelatonin and core flowering regulators, namely DELLA proteins, ROS, and phytohormones. Further efforts are needed to address some intriguing aspects, such as the exploration of the association between phytomelatonin and photoperiodic flowering, phytomelatonin-related floral MADS-box genes, the crosstalk between phytomelatonin and phytohormones, as well as its potential applications in agriculture.

Inhibition of soluble epoxide hydrolase attenuates a high-fat diet-mediated renal injury by activating PAX2 and AMPK.

A high-fat diet (HFD) causes obesity-associated morbidities involved in macroautophagy and chaperone-mediated autophagy (CMA). AMPK, the mediator of macroautophage, has been reported to be inactivated in HFD-caused renal injury. However, PAX2, the mediator for CMA, has not been reported in HFD-caused renal injury. Here we report that HFD-caused renal injury involved the inactivation of Pax2 and Ampk, and the activation of soluble epoxide hydrolase (sEH), in a murine model. Specifically, mice fed on an HFD for 2, 4, and 8 wk showed time-dependent renal injury, the significant decrease in renal Pax2 and Ampk at both mRNA and protein levels, and a significant increase in renal sEH at mRNA, protein, and molecular levels. Also, administration of an sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea, significantly attenuated the HFD-caused renal injury, decreased renal sEH consistently at mRNA and protein levels, modified the renal levels of sEH-mediated epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) as expected, and increased renal Pax2 and Ampk at mRNA and/or protein levels. Furthermore, palmitic acid (PA) treatment caused significant increase in Mcp-1, and decrease in both Pax2 and Ampk in murine renal mesangial cells (mRMCs) time- and dose-dependently. Also, 14(15)-EET (a major substrate of sEH), but not its sEH-mediated metabolite 14,15-DHET, significantly reversed PA-induced increase in Mcp-1, and PA-induced decrease in Pax2 and Ampk. In addition, plasmid construction revealed that Pax2 may positively regulate Ampk transcriptionally in mRMCs. This study provides insights into and therapeutic target for the HFD-mediated renal injury.

Solanum steroidal glycoalkaloids: structural diversity, biological activities, and biosynthesis.

Covering: up to 1 October 2020Solanum steroidal glycoalkaloids (SGA), characterized by nitrogenous steroidal aglycone and glycoside residues, mainly occur in the Solanum species, including economically important edible plants such as potato, tomato, and eggplant. To date, 107 SGA assigned to six total skeletons have been identified from Solanum plants. SGA have unique structures and display significant pharmacological activities such as cytotoxic, antimicrobial, anticholesterol, and some are well-known poisons. The biosynthesis pathway, transcriptional regulation, and the evolution of SGA are also examined in detail. This report updates the chemical knowledge of the naturally occurring SGA from Solanum species, thereby providing an in-depth analysis of their diversity, biological activities, and biosynthesis.

Melatonin confers heavy metal-induced tolerance by alleviating oxidative stress and reducing the heavy metal accumulation in Exophiala pisciphila, a dark septate endophyte (DSE).

BACKGROUND: Melatonin (MT), ubiquitous in almost all organisms, functions as a free radical scavenger. Despite several reports on its role as an antioxidant in animals, plants, and some microorganisms, extensive studies in filamentous fungi are limited. Based upon the role of melatonin as an antioxidant, we investigated its role in heavy metal-induced stress tolerance in Exophiala pisciphila, a dark septate endophyte (DSE), by studying the underlying mechanisms in alleviating oxidative stress and reducing heavy metal accumulation. RESULTS: A significant decrease in malondialdehyde (MDA) and oxygen free radical (OFR) in E. pisciphila was recorded under Cd, Zn, and Pb stresses as compared to the control. Pretreatment of E. pisciphila with 200.0 µM exogenous melatonin significantly increased the activity of superoxide dismutase (SOD) under Zn and Pb stresses. Pretreatment with 200.0 µM melatonin also lowered Cd, Zn, and Pb concentrations significantly. Melatonin production was enhanced by Cd, Cu, and Zn after 2 d, and melatonin biosynthetic enzyme genes, E. pisciphila tryptophan decarboxylase (EpTDC1) and serotonin N-acetyltransferase (EpSNAT1), were transcriptionally upregulated. The overexpression of EpTDC1 and N-acetylserotonin O-methyltransferase (EpASMT1) in Escherichia coli and Arabidopsis thaliana enhanced its heavy metal-induced stress tolerance. The overexpression of EpTDC1 and EpASMT1 reduced the Cd accumulation in the whole A. thaliana plants, especially in the roots. CONCLUSIONS: Melatonin conferred heavy metal-induced stress tolerance by alleviating oxidative stress, activating antioxidant enzyme SOD, and reducing heavy metal accumulation in E. pisciphila. Melatonin biosynthetic enzyme genes of E. pisciphila also played key roles in limiting excessive heavy metal accumulation in A. thaliana. These findings can be extended to understand the role of melatonin in other DSEs associated with economically important plants and help develop new strategies in sustainable agriculture practice where plants can grow in soils contaminated with heavy metals.

Melatonin inhibits seed germination by crosstalk with abscisic acid, gibberellin, and auxin in Arabidopsis.

Seed germination, an important developmental stage in the life cycle of seed plants, is regulated by complex signals. Melatonin is a signaling molecule associated with seed germination under stressful conditions, although the underlying regulatory mechanisms are largely unknown. In this study, we showed that a low concentration (10 µM or 100 µM) of melatonin had no effect on seed germination, but when the concentration of melatonin increased to 500 µM or 1000 µM, seed germination was significantly inhibited in Arabidopsis. RNA sequencing analysis showed that melatonin regulated seed germination correlated to phytohormones abscisic acid (ABA), gibberellin (GA), and auxin. Further investigation revealed that ABA and melatonin synergistically inhibited seed germination, while GA and auxin antagonized the inhibitory effect of seed germination by melatonin. Disruption of the melatonin biosynthesis enzyme gene serotonin N-acetyltransferase (SNAT) or N-acetylserotonin methyltransferase (ASMT) promoted seed germination, while overexpression of ASMT inhibited seed germination. Taken together, our study sheds new light on the function and mechanism of melatonin in modulating seed germination in Arabidopsis.

Melatonin synthesis genes N-acetylserotonin methyltransferases evolved into caffeic acid O-methyltransferases and both assisted in plant terrestrialization.

Terrestrialization is one of the most momentous events in the history of plant life, which leads to the subsequent evolution of plant diversity. The transition species, in this process, had to acquire a range of adaptive mechanisms to cope with the harsh features of terrestrial environments compared to that of aquatic habitat. As an ancient antioxidant, a leading regulator of ROS signaling or homeostasis, and a presumed plant master regulator, melatonin likely assisted plants transition to land and their adaption to terrestrial ecosystems. N-acetylserotonin methyltransferases (ASMT) and caffeic acid O-methyltransferases (COMT), both in the O-methyltransferase (OMT) family, catalyze the core O-methylation reaction in melatonin biosynthesis. How these two enzymes with close relevance evolved in plant evolutionary history and whether they participated in plant terrestrialization remains unknown. Using combined phylogenetic evidence and protein structure analysis, it is revealed that COMT likely evolved from ASMT by gene duplication and subsequent divergence. Newly emergent COMT gained a significantly higher ASMT activity to produce greater amounts of melatonin for immobile plants to acclimate to the stressful land environments after evolving from the more environmentally-stable aquatic conditions. The COMT genes possess more conserved substrate-binding sites at the amino acid level and more open protein conformation compared to ASMT, and getting a new function to catalyze the lignin biosynthesis. This development directly contributed to the dominance of vascular plants among the Earth's flora and prompted plant colonization of land. Thus, ASMT, together with its descendant COMT, might play key roles in plant transition to land. The current study provides new insights into plant terrestrialization with gene duplication contributing to this process along with well-known horizontal gene transfer.

Aconitum Diterpenoid Alkaloid Profiling to Distinguish between the Official Traditional Chinese Medicine (TCM) Fuzi and Adulterant Species Using LC-qToF-MS with Chemometrics.

The lateral roots of Aconitum carmichaelii, known in Chinese as fuzi, are officially recognized as a materia medica in the Chinese Pharmacopoeia and used culinarily to prepare herbal soups. A strategy combining UPLC-qToF-MS analysis of A. carmichaelii and its intraspecies and interspecies chemometrics study was developed to examine the distribution of Aconitum marker metabolites. Four diterpenoid alkaloids were recognized to be important markers in fuzi, and another 15 markers were identified to differentiate A. carmichaelii from adulterant species. The detected fuzi markers, mesaconitine (47) and hypaconitine (51), are known to be the principal toxins in this herb, while fuziline (6) and benzoylmesaconine (25) are associated with its medicinal properties. Additional marker compounds have been detected in other Aconitum species that are useful for identifying adulteration. This study provides a useful resource for detecting traditional Chinese medicine (TCM) adulterants and assisting in the quality control of botanical products in TCM and beyond.

Structural diversity, bioactivities, and biosynthesis of natural diterpenoid alkaloids.

Covering: 2009 to 2018. Diterpenoid alkaloids, originating from the amination of natural tetracyclic diterpenes, are a diverse class of compounds having complex structural features with many stereocenters. The important pharmacological activities and structural complexity of the diterpenoid alkaloids have long interested scientists due to their medicinal uses, infamous toxicity, and unique biosynthesis. Since 2009, 373 diterpenoid alkaloids, assigned to 46 skeletons, have been isolated and identified from plants mostly in the Ranunculaceae family. The names, classes, molecular weight, molecular formula, NMR data, and plant sources of these diterpene alkaloids are collated here. This review will be a detailed update of the naturally occurring diterpene alkaloids reported from the plant kingdom from 2009-2018, providing an in-depth discussion of their diversity, biological activities, pharmacokinetics, toxicity, application, evolution, and biosynthesis.

Conosiligins A-D, Ring-Rearranged Tremulane Sesquiterpenoids from Conocybe siliginea.

Conosiligins A-D (1-4), four ring-rearranged sesquiterpenoids, were isolated from cultures of the basidiomycete Conocybe siliginea. Their structures and absolute configurations were determined by detailed spectroscopic analyses and equivalent circulating density (ECD) calculations. Compounds 1 and 2 possess a 5/8-fused ring system, while 3 has a 5/6-fused backbone conjugated with a γ-lactone. Compound 4 is a 5,6-seco tremulane derivative with the loss of a skeletal carbon, featuring a tetracyclic system involving a pyranone moiety. Compounds 3 and 4 inhibited Con A-induced T cell proliferation with IC50 values of 12.3 and 6.6 µM, respectively.

Fungi isolated from host protocorms accelerate symbiotic seed germination in an endangered orchid species (Dendrobium chrysotoxum) from southern China.

To ensure long-term survival of epiphytic orchids through active reintroduction, more research on critical life cycle stages such as seed germination and seedling establishment are needed. In this study, we used in vitro germination experiments to investigate the role of mycorrhizal fungi in determining seed germination and growth in the endangered epiphytic orchid species, Dendrobium chrysotoxum. Symbiotic seed germination experiments were conducted for 90 days under different light conditions with fungal strains isolated from protocorms of D. chrysotoxum and three sister species. Molecular analyses showed that five strains belonged to the typical orchid mycorrhizal family Tulasnellaceae, whereas the other two strains belonged to the Sebacinaceae and the genus Coprinellus. Fungal inoculation, light conditions, and their interaction had a significant effect on protocorm formation and seedling development. Three fungal isolates, including two from D. chrysotoxum and one from D. catenatum, significantly stimulated protocorm formation and seedling development under light conditions. However, fungi isolated from host protocorms (GC-14 and GC-15) produced the highest number of seedlings after 50 days (49.5 ± 8.5%, 51.3 ± 9.0%, respectively), while the fungus isolated from D. catenatum protocorms produced the maximum number of seedlings only after 90 days (48.7 ± 16.1%). To conclude, this study has shown that light conditions and the identity of fungi had a strong effect on in vitro seed germination and seedling formation in an epiphytic orchid, with fungi isolated from host protocorms leading to accelerated germination and seedling formation. Therefore, fungal source should be taken into account when using seeds and compatible fungi for seedling propagation and in situ reintroduction.

Two new bis-C20-diterpenoid alkaloids with anti-inflammation activity from Aconitum bulleyanum.

Two new bis-C20-diterpenoid alkaloids, bulleyanines A and B (1 and 2), were isolated from Aconitum bulleyanum. Their structures were elucidated by comprehensive spectroscopic analyses including UV, IR, MS, 1D and 2D NMR. Biological activity tests indicated that compound 1 exhibited significant inhibitory activity against nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW264.7 macrophages with the inhibition rate of 74.60% (40 µmol/L), while positive control dexamethasone gave 78.70% inhibition at 100 µg/ml.

Four new C19-diterpenoid alkaloids from the roots of Aconitum ouvrardianum.

Four new C19-diterpenoid alkaloids, 3-dehydroxyl-lipoindaconitine (1), 8-dehydroxyl-bikhaconine (2), 19R-acetonyl-talatisamine (3), and 16-hydroxyl-vilmorisine (4), were isolated from the roots of Aconitum ouvrardianum. Their structures were elucidated by spectral analyses, including ESI-MS, HR-ESI-MS, IR, UV, 1D and 2D NMR.

Overexpression of the Melatonin Synthesis-Related Gene SlCOMT1 Improves the Resistance of Tomato to Salt Stress.

Melatonin can increase plant resistance to stress, and exogenous melatonin has been reported to promote stress resistance in plants. In this study, a melatonin biosynthesis-related SlCOMT1 gene was cloned from tomato (Solanum lycopersicum Mill. cv. Ailsa Craig), which is highly expressed in fruits compared with other organs. The protein was found to locate in the cytoplasm. Melatonin content in SlCOMT1 overexpression transgenic tomato plants was significantly higher than that in wild-type plants. Under 800 mM NaCl stress, the transcript level of SlCOMT1 in tomato leaf was positively related to the melatonin contents. Furthermore, compared with that in wild-type plants, levels of superoxide and hydrogen peroxide were lower while the content of proline was higher in SlCOMT1 transgenic tomatoes. Therefore, SlCOMT1 was closely associated with melatonin biosynthesis confers the significant salt tolerance, providing a clue to cope with the growing global problem of salination in agricultural production.

Enriched networks 'nucleoside/nucleotide and ribonucleoside/ribonucleotide metabolic processes' and 'response to stimulus' potentially conferred to drought adaptation of the epiphytic orchid Dendrobium wangliangii.

Dendrobium wangliangii is an endangered and epiphytic orchid with tolerance to seasonally extreme arid conditions and occurs exclusively in the hot-dry valley area of southwestern China. To reveal its molecular basis responsible for ecological adaptation, large-scale transcriptome sequencing was performed using Illumina sequencing with pooled mRNA extracted from whole plants and pseudobulbs during drought and rainy seasons. Based on the target transcript selection, the differentially expressed genes were related to 8 well-known drought-tolerant categories, and to morphological traits in resistance to water stress including pseudobulbs and roots. Further gene ontology enrichment analysis revealed that 'nucleoside/nucleotide and ribonucleoside/ribonucleotide metabolic processes' and 'response to stimulus' were the two most important aspects in resistance to drought stress with respect to the whole plant. In addition, the difference in the number and category of differentially expressed genes in whole plant and stem suggested the involvement of genes specifically localized in the stem, such as GTP-binding protein, lipases, signaling related transcripts and those involved in the ATP metabolic process. The comprehensive analysis of the epiphytic orchid in response to water deprivation indicates that integral tactics lead to active adaptation as a basal defense response to drought stress by the endangered epiphyte, including the collaboration of metabolic processes, responses to a various stimulus and other candidate genes contribute to its extreme drought tolerance. Insights from this study can be further utilized to understand stress-responsive genes in other medicinally important species and to improve the drought tolerance of food crops.

Identification of Potential Biomarkers from Aconitum carmichaelii, a Traditional Chinese Medicine, Using a Metabolomic Approach.

Despite their well-known toxicity, Aconitum species are important traditional medicines worldwide. Aconitum carmichaelii, known in Chinese as (fuzi), is an officially recognized traditional Chinese medicine with characteristic analgesic and anti-inflammatory activities, whose principal pharmacological ingredients are considered as aconitine-type diterpene alkaloids. Notwithstanding the long-recorded use of A. carmichaelii in traditional Chinese medicine, no single-entity aconitum alkaloid drug has been developed for clinical use. UPLC-Q-TOF-MS was used to investigate the marker compounds that can be used to differentiate A. carmichaelii from seven other Aconitum species collected in Yunnan Province. Nontargeted principle component analysis scores plots found that all the tested Aconitum species clustered into three distinct groups, and A. carmichaelii was significantly different chemically than the other seven species. Furthermore, the primary and lateral roots of A. carmichaelii also showed significant differences. Using orthogonal partial least squares discriminate analysis analysis, eight marker compounds were identified, including 14-acetylkarakoline, aconitine, carmichaeline, fuziline, hypaconitine, mesaconitine, neoline, and talatisamine. Four of these aconitum alkaloids, fuziline, hypaconitine, mesaconitine, and neoline, showed significant analgesic activity in a dose-dependent manner compared to the negative and positive controls. However, hypaconitine, mesaconitine, and neoline exhibited significant acute toxicity activity, while fuziline showed no acute toxicity in mice, suggesting the relative safety of this alkaloid. This study provides a good example of how to differentiate an authentic medicinal plant from common adulterants using a metabolomics approach, and to identify compounds that may be developed into new drugs.

The Role of Phyto-Melatonin and Related Metabolites in Response to Stress.

Plant hormone candidate melatonin has been widely studied in plants under various stress conditions, such as heat, cold, salt, drought, heavy metal, and pathogen attack. Under stress, melatonin usually accumulates sharply by modulating its biosynthesis and metabolic pathways. Beginning from the precursor tryptophan, four consecutive enzymes mediate the biosynthesis of tryptamine or 5-hydroxytryptophan, serotonin, N-acetylserotonin or 5-methoxytryptamine, and melatonin. Then, the compound is catabolized into 2-hydroxymelatonin, cyclic-3-hydroxymelatonin, and N¹-acetyl-N²-formyl-5-methoxyknuramine through 2-oxoglutarate-dependent dioxygenase catalysis or reaction with reactive oxygen species. As an ancient and powerful antioxidant, melatonin directly scavenges ROS induced by various stress conditions. Furthermore, it confreres stress tolerance by activating the plant's antioxidant system, alleviating photosynthesis inhibition, modulating transcription factors that are involved with stress resisting, and chelating and promoting the transport of heavy metals. Melatonin is even proven to defense against pathogen attacks for the plant by activating other stress-relevant hormones, like salicylic acid, ethylene, and jasmonic acid. Intriguingly, other precursors and metabolite molecules involved with melatonin also can increase stress tolerance for plant except for unconfirmed 5-methoxytryptamine, cyclic-3-hydroxymelatonin, and N¹-acetyl-N²-formyl-5-methoxyknuramine. Therefore, the precursors and metabolites locating at the whole biosynthesis and catabolism pathway of melatonin could contribute to plant stress resistance, thus providing a new perspective for promoting plant stress tolerance.

Two new C19-diterpenoid alkaloids from Aconitum tsaii.

Two new C19-diterpenoid alkaloids, 14-benzoylliljestrandisine (1) and 14-anisoylliljestrandisine (2), were isolated from the roots of Aconitum tsaii. Their structures were elucidated by different spectroscopic (IR, UV, 1D and 2D NMR) and mass-spectrometric techniques.

Genomic Analysis of the ASMT Gene Family in Solanum lycopersicum.

Acetylserotonin methyltransferase (ASMT) is the last enzyme of melatonin biosynthesis and may play a rate-limiting role in the melatonin production of plants. In this study, systematic analysis of the ASMT gene family in tomato (Solanum lycopersicum Mill) has been presented by the integration of the structural features, phylogenetic relationships, exon/intron configuration, and expression profile during growth and development, as well as biotic stresses. The results revealed that the tomato genome encoded a minimum of 14 members, containing three probable encoded pseudogenes. Chromosome mapping indicated that the family had probably expanded via tandem duplication events. Genome-wide RNA-seq and qRT-PCR based gene expression analysis revealed that almost half of the SlASMT genes were expressed in at least one of the experimental stages studied and also showed differential accumulation. Furthermore, the tandem duplicated SlASMT genes showed differential expression levels, which indicated probable functional divergence during the course of the evolution. Finally, this study also determined that some SlASMT genes were induced by multiple pathogens. The results suggested that these genes could be involved in tomato plant response to biotic stresses.