Selection and Validation of Reference Genes for Gene Expression Studies in Euonymus japonicus Based on RNA Sequencing.

Euonymus japonicus is one of the most low-temperature-tolerant evergreen broad-leaved tree species in the world and is widely used in urban greening. However, there are very few molecular biology studies on its low-temperature tolerance mechanism. So far, no researcher has selected and reported on its reference genes. In this study, 21 candidate reference genes (12 traditional housekeeping genes and 9 other genes) were initially selected based on gene expression and coefficient of variation (CV) through RNA-Seq (unpublished data), and qRT-PCR was used to detect the expression levels of candidate reference genes in three different groups of samples (leaves under different temperature stresses, leaves of plants at different growth stages, and different organs). After further evaluating the expression stability of these genes using geNorm, NormFinder, Bestkeeper, and RefFind, the results show that the traditional housekeeping gene eIF5A and the new reference gene RTNLB1 have good stability in the three different groups of samples, so they are reference genes with universality. In addition, we used eIF5A and RTNLB1 as reference genes to calibrate the expression pattern of the target gene EjMAH1, which confirmed this view. This article is the first to select and report on the reference gene of E. japonicus, laying the foundation for its low-temperature tolerance mechanism and other molecular biology research.

Mechanism of Rhodiola rosea-Euonymus alatus drug pair against rheumatoid arthritis: Network pharmacology and experimental validation.

PURPOSE: The present study aimed to explore the potential components and mechanisms of Rhodiola rosea-Euonymus alatus drug pair (TY) that ameliorate rheumatoid arthritis (RA). METHODS: The main active components, core targets, and important pathways of TY against RA were predicted by network pharmacology analysis. The binding activity between the main active components and the core targets was verified by the molecular docking technique. Collagen-induced arthritis (CIA) rat model and tumor necrosis factor (TNF)-α-induced fibroblast-like synovial cells in human RA (HFLS-RA) model were established, respectively. The core targets were verified by cell counting kit-8 assay, hematoxylin eosin, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and Western blot analysis, and the therapeutic effect of TY was evaluated. RESULTS: A total of 18 possible components and 34 core targets were obtained by network pharmacology, among which inflammatory response, phosphatidylinositide 3-kinases (PI3K)-AKT and MAPK pathways were involved in the therapeutic effect of TY on RA. The results of molecular docking showed that kaempferol and quercetin had high binding affinity to interleukin (IL)-1ß, IL-6, matrix metalloproteinase (MMP)9, and TNF-α. In vivo and in vitro experiments showed that TY dose-dependently inhibited the proliferation of HFLS-RA cells induced by TNF-α, and significantly reduced the paw swelling and arthritis scores in CIA rats. At the same time, TY inhibited the production of inflammatory factors in CIA rat serum and TNF-α-induced HFLS-RA cells. It also decreased the expression of PI3K, phospho-protein kinase B, MMP1, MMP3, MMP9, and increased the protein and mRNA levels of tissue inhibitors of MMPs (TIMP)1 in synovial tissue. CONCLUSION: TY can inhibit the PI3K/AKT signaling pathway and regulate the balance between MMPs and TIMP, thus playing a therapeutic role in RA.

Instantaneous sap flow velocity simulation of Euonymus bungeanus based on neural network optimization model.

The sap flow of trees is complex and difficult to express with multivariate linear or empirical models. A simple and feasible method on the basis of understanding sap flow variation to simulate its variation with environmental factors is of special importance for quantitatively analyzing forest ecohydrological processes and regional water demand. In this study, with one of the shelter forest species Euonymus bungeanus in the east sandy land of Yellow River in Ningxia as the research object, we continuously measured the trunk sap flow velocity by thermal diffusion sap flow meter, and analyzed the effects of environmental factors on stem sap flow. We used the particle swarm optimization (PSO) and sparrow search algorithm (SSA) optimized neural network model to predict sap flow velocity of E. bungeanus. Results showed that the main environmental factors influencing sap flow were solar radiation, vapor pressure deficit, air temperature, and relative humidity, with the influencing importance of 32.5%, 25.3%, 22.0% and 16.1%, respectively. The response process between sap flow and environmental factors presented a hysteresis loop relationship. The optimized BP, Elman and ELM neural network models improved the comprehensive evaluation index (GPI) by 1.5%, 30.0% and 5.3%, respectively. Compared with the PSO-Elman and SSA-ELM optimization models, the SSA-BP optimization model had the best prediction results with an improvement of 1.0% and 23.2% in GPI, respectively. Therefore, the prediction results of the BP neural network model based on the sparrow search algorithm could be used as an optimal model for predicting instantaneous sap flow velocity of E. bungeanus.

Adaptation to pollination by fungus gnats underlies the evolution of pollination syndrome in the genus Euonymus.

BACKGROUND AND AIMS: Dipteran insects are known pollinators of many angiosperms, but knowledge on how flies affect floral evolution is relatively scarce. Some plants pollinated by fungus gnats share a unique set of floral characters (dark red display, flat shape and short stamens), which differs from any known pollination syndromes. We tested whether this set of floral characters is a pollination syndrome associated with pollination by fungus gnats, using the genus Euonymus as a model. METHODS: The pollinator and floral colour, morphology and scent profile were investigated for ten Euonymus species and Tripterygium regelii as an outgroup. The flower colour was evaluated using bee and fly colour vision models. The evolutionary association between fungus gnat pollination and each plant character was tested using a phylogenetically independent contrast. The ancestral state reconstruction was performed on flower colour, which is associated with fungus gnat pollination, to infer the evolution of pollination in the genus Euonymus. KEY RESULTS: The red-flowered Euonymus species were pollinated predominantly by fungus gnats, whereas the white-flowered species were pollinated by bees, beetles and brachyceran flies. The colour vision analysis suggested that red and white flowers are perceived as different colours by both bees and flies. The floral scents of the fungus gnat-pollinated species were characterized by acetoin, which made up >90 % of the total scent in three species. Phylogenetically independent contrast showed that the evolution of fungus gnat pollination is associated with acquisition of red flowers, short stamens and acetoin emission. CONCLUSIONS: Our results suggest that the observed combination of floral characters is a pollination syndrome associated with the parallel evolution of pollination by fungus gnats. Although the role of the red floral display and acetoin in pollinator attraction remains to be elucidated, our finding underscores the importance of fungus gnats as potential contributors to floral diversification.

Integrated metabolomics and transcriptomics analysis reveals that the change of apoplast metabolites contributes to adaptation to winter freezing stress in Euonymus japonicus.

Euonymus japonicus, a common urban street tree, can withstand winter freezing stress in temperate regions. The apoplast is the space outside the plasma membrane, and the changes of metabolites in apoplast may be involved in plant adaptation to adverse environments. To reveal the molecular mechanism underlying the winter freezing stress tolerance in E. japonicus, the changes in physiological and biochemical indexes, apoplast metabolites, and gene expression in the leaves of E. japonicus in early autumn and winter were analyzed. A total of 300 differentially accumulated metabolites were identified in apoplast fluids in E. japonicus, which were mainly related to flavone and flavonol biosynthesis, and galactose metabolism, amino acid synthesis, and unsaturated fatty acid synthesis. Integrated metabolomics and transcriptomics analysis revealed that E. japonicus adjust apoplast metabolites including flavonoids such as quercetin and kaempferol, and oligosaccharides such as raffinose and stachyose, to adapt to winter freezing stress through gene expression regulation. In addition, the regulation of ABA and SA biosynthesis and signal transduction pathways, as well as the activation of the antioxidant enzymes, also played important roles in the adaptation to winter freezing stress in E. japonicus. The present study provided essential data for understanding the molecular mechanism underlying the adaptation to winter freezing stress in E. japonicus.

Euonymus sachalinensis Induces Apoptosis by Inhibiting the Expression of c-Myc in Colon Cancer Cells.

We hypothesized that Euonymus sachalinensis (ES) induces apoptosis by inhibiting the expression of c-Myc in colon cancer cells, and this study proved that the methanol extract of ES has anticancer effects in colon cancer cells. ES belongs to the Celastraceae family and is well known for its medicinal properties. Extracts of species belonging to this family have been used to treat diverse diseases, including rheumatoid arthritis, chronic nephritis, allergic conjunctivitis, rhinitis, and asthma. However, ES has been targeted because there are currently few studies on the efficacy of ES for various diseases, including cancer. ES lowers cell viability in colon cancer cells and reduces the expression of c-Myc protein. We confirm that the protein level of apoptotic factors such as PARP and Caspase 3 decrease when ES is treated with Western blot, and confirm that DNA fragments occur through TUNEL assay. In addition, it is confirmed that the protein level of oncogenes CNOT2 and MID1IP1 decrease when ES is treated. We have also found that ES enhances the chemo-sensitivity of 5-FU in 5-FU-resistant cells. Therefore, we confirm that ES has anticancer effects by inducing apoptotic cell death and regulating the oncogenes CNOT2 and MID1IP1, suggesting its potential for use in the treatment of colon cancer.

Influence of exposure history on the particle retention capacity and physiological responses of Euonymus japonicus Thunb. var. aurea-marginatus Hort.

Green plants in urban environments experience cyclical particulate matter stress. And this history of exhaust exposure may generate stress memory in plants, which may alter their subsequent response. Studies combining urban pollution characteristics and stress memory are limited. Therefore, we selected E. japonicus var. aurea-marginatus, a common urban greening tree species in the Yangtze River Delta, and conducted an experiment in three periods: the initial pollution period (S1: 28 days), the recovery period (R: 14 days) and the secondary pollution period (S2: 28 days). The experimental design consisted of an elevated pollution treatment (173 µg•cm-3) and an ambient control (34 µg•cm-3) with three replicates. In S2, the net total particle retention and saturated particle retention decreased by 11.5% and 19.3%, respectively, while PM10 and PM2.5 did not change significantly. E. japonicus var. aurea-marginatus exhibited recovery of chlorophyll levels, slower degradation of carotenoid, faster accumulation of ASA, lower accumulation of MDA, reduced activity of SOD under the second pollution period, and the period had a significant effect on the physiological indicators. Collectively, the effect of autoexhaust exposure history on the particle retention capacity of selected plant varied across particle sizes, and stress memory may confer plant resistance to recurrent exhaust pollution via combined regulations of physiological responses. Fine particles which pose a great risk to human health arise predominantly from vehicular traffic and energy production. So, E. japonicus tends to play a stabilising role in particle retention in industrial, traffic and residential areas.

Characterization of the Complete Chloroplast Genome and Phylogenetic Implications of Euonymus microcarpus (Oliv.) Sprague.

Euonymus microcarpus (Oliv.) Sprague, is a species of evergreen shrub of the genus Euonymus, family Celastraceae. Here, we extracted the genomic DNA from the leaves of E. microcarpus and constructed a paired-end library. The chloroplast genome of E. microcarpus was generated with the high-throughput sequencing by the illumina Hiseq X Ten platform and de novo assembly. The chloroplast genome had a quadripartite structure, containing a long single copy region with a size of 85,386 bp and a short single copy region with a size of 18,456 bp, separated by two inverted repeat regions of 26,850 bp. The chloroplast genome contained 133 genes identified in total, including 87 potential protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A total of 282 simple sequence repeats and 63 long repeats were found. Furthermore, the phylogenetic relationships inferred that E. microcarpus is sister to E. japonicus and E. schensianus. A comparison of the structure of the chloroplast genomes of eight Euonymus species suggests a nucleotide variability of the junction sites and a higher divergence of non-coding regions, compared to the coding regions. The original findings of the study serves as a good reference for chloroplast genome assembly and a valuable foundation for the genetic diversity and evolution of E. microcarpus.

Analysis of the complete plastomes and nuclear ribosomal DNAs from Euonymus hamiltonianus and its relatives sheds light on their diversity and evolution.

Euonymus hamiltonianus and its relatives (Celastraceae family) are used for ornamental and medicinal purposes. However, species identification in Euonymus is difficult due to their morphological diversity. Using plastid genome (plastome) data, we attempt to reveal phylogenetic relationship among Euonymus species and develop useful markers for molecular identification. We assembled the plastome and nuclear ribosomal DNA (nrDNA) sequences from five Euonymus lines collected from South Korea: three Euonymus hamiltonianus accessions, E. europaeus, and E. japonicus. We conducted an in-depth comparative analysis using ten plastomes, including other publicly available plastome data for this genus. The genome structures, gene contents, and gene orders were similar in all Euonymus plastomes in this study. Analysis of nucleotide diversity revealed six divergence hotspots in their plastomes. We identified 339 single nucleotide polymorphisms and 293 insertion or deletions among the four E. hamiltonianus plastomes, pointing to abundant diversity even within the same species. Among 77 commonly shared genes, 9 and 33 were identified as conserved genes in the genus Euonymus and E. hamiltonianus, respectively. Phylogenetic analysis based on plastome and nrDNA sequences revealed the overall consensus and relationships between plastomes and nrDNAs. Finally, we developed six barcoding markers and successfully applied them to 31 E. hamiltonianus lines collected from South Korea. Our findings provide the molecular basis for the classification and molecular taxonomic criteria for the genus Euonymus (at least in Korea), which should aid in more objective classification within this genus. Moreover, the newly developed markers will be useful for understanding the species delimitation of E. hamiltonianus and closely related species.

Intervention effect of Potentilla discolor-Euonymus alatus on intestinal flora of type 2 diabetes mellitus rats.

OBJECTIVE: With this study, we aimed at exploring the regulation mechanism of Potentilla discolor-Euonymus alatus on intestinal flora of T2DM (Type 2 Diabetes Mellitus) rats induced by high-fat diet combined with streptozotocin. MATERIALS AND METHODS: T2DM rats were induced by high-fat diet combined with streptozotocin. There were normal control group, model group, metformin group, high-dose Chinese medicine group and low-dose Chinese medicine group. Each group included 10 rats. Normal control group: normal feeding, no modeling, ordinary feed, and gavage of 0.9% normal saline. Model group: T2DM rats, high-fat diet, and gavage of 0.9% normal saline. Metformin group: T2DM rats, high-fat diet and fed with metformin solution. High-dose Chinese medicine group: T2DM rats, high-fat diet, and gavage of concentrated Chinese medicine at a dose of 6 times the clinical dose. Low-dose Chinese medicine group: T2DM rats, high-fat diet, and gavage of concentrated Chinese medicine at a dose twice the clinical dose. The general situation of T2DM rats was observed, and the changes of intestinal flora were observed with 16SrDNA sequencing. RESULTS: The T2DM rats induced by high-fat diet combined with streptozotocin were molded. After intervention, at the class level, the ratio of γ-proteobacteria was 22.30% in the model group, 11.97% in the metformin group, 3.24% in the high-dose Chinese herbs group and 1.72% in the low-dose Chinese herbs group; the ratio of Erysipelothrix insidiosa was 4.73% in the model group, 4.68% in the metformin group, 3.93% in the high-dose Chinese herbsgroup and 2.92% in the low dose group; the ratio of Lactinobacillus was 2.30% in the model group, 0.01% in the metformin group, 0.00% in the high-dose Chinese herbs group, and 0.00% low-dose Chinese herbs group; at the portal level, the Firmicutes/Bacteroides was 0.88 in the normal control group, 3.40 in the model group, 1.71 in the metformin group, 2.74 in high-dose Chinese medicine group, and 1.34 in low-dose Chinese medicine group; at the genus level, the relative abundance of Lactobacillus in the model group was 3.28%, that of Akkermansia was 1.99%, that of Shigella coli was 22.08%, and that of Vibrio phaseus was 7.67%. All of them were improved after the intervention of metformin and traditional Chinese medicine. CONCLUSIONS: Potentilla discolor-Euonymus Alatus could improve the composition and structure of intestinal flora in T2DM rats and regulate the diversity of intestinal flora. The ratio of Firmicutes/Bacteroidetes was adjusted, mainly to increase the number of Bacteroides; the flora related to intestinal barrier was adjusted, mainly to increase the number of Lactobacillus and Akkermansia bacteria.

Euonymus alatus Twig Extract Protects against Scopolamine-Induced Changes in Brain and Brain-Derived Cells via Cholinergic and BDNF Pathways.

In the current study, the therapeutic and preventive effects of Euonymus alatus (EA) twig extract were investigated in a mouse model of cognitive deficit and B35 cells. Twig extract 1 was extracted with 70% ethanol and later twig extract 2 was extracted through liquid-liquid extraction with 70% ethanol and hexane. EA twig 2 (300 mg/kg) along with the standard drug donepezil (5 mg/kg) were orally administered to the mice for 34 days. Scopolamine was given intraperitoneally for 7 days. Administration of EA twig extract 2 significantly improved the passive avoidance test (PAT) in mice. EA twigs extract also restored the scopolamine-reduced brain-derived neurotrophic factor (BDNF)/extracellular regulated kinase (ERK)/cyclic AMP responsive element binding protein (CREB) signaling in B35 cells and the mouse hippocampus. In addition, EA twig extract significantly inhibited the acetylcholine esterase (AChE) activity in B35 cells in a dose-dependent manner. Chromatography and ESI MS analysis of EA twig extract revealed the presence of flavonoids; epicatechin, taxifolin, aromadendrin, and naringenin with catechin being the most abundant. These flavonoids exerted protective effects alone and had the possibility of synergistic effects in combination. Our work unmasks the ameliorating effect of EA twig extract 2 on scopolamine-associated cognitive impairments through the restoration of cholinergic systems and the BDNF/ERK/CREB pathway.

The water extracts of Euonymus alatus (Thunb.) Siebold attenuate diabetic retinopathy by mediating angiogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Euonymus alatus (Thunb.) Siebold (family Celastraceae) is a deciduous woody shrub that is recorded in ShenNong BenCaoJing. It has been widely used for diabetes in traditional Chinese medicine. AIM OF THE STUDY: This study aimed to identify the most effective extract of Euonymus alatus (EA) against high glucose-induced endothelial cells in vitro, evaluate its pharmacological effect on retinopathy in diabetic mice and explore its underlying mechanism by RNA sequencing. METHODS: Retinal vascular endothelial cells (RF/6A) were treated with normal glucose (5.5 mmol/L glucose), high glucose (25 mmol/L glucose) or high glucose plus methanol extracts of EA (MEA), ethyl acetate extracts of EA (EEA) or water extracts of EA (WEA). The cytotoxicity and cell viability were determined by Cell Counting Kit-8 (CCK-8) assay. Cell migration was examined using the Transwell assay, and tube formation ability was measured using the Matrigel assay. Then, the KK-Ay mice were administered WEA or water for 12 weeks. The velocities of ocular blood flow were determined by Doppler ultrasound. RNA sequencing and reverse transcription quantitative PCR (RT-qPCR) were performed on WEA-stimulated RF/6A cells to reveal the underlying mechanism. RESULTS: The cytotoxicity assay found that 30 µg/mL MEA, 20 µg/mL EEA and 30 µg/mL WEA had no toxic effect on RF/6A cells. The cell viability results showed that MEA, EEA and WEA all decreased cell viability. Compared with the high-glucose group, both MEA and WEA decreased the number of migrated cells, while the inhibition rate of WEA was higher. The Matrigel results showed that 30 µg/mL WEA effectively reduced the total tube length. Moreover, WEA improved the haemodynamics of the central retinal artery. RNA sequencing coupled with RT-qPCR verified that WEA regulated angiogenesis-related factors in high glucose-stimulated RF/6A cells. CONCLUSIONS: WEA inhibits the migration and tube formation of RF/6A cells and improves diabetic retinopathy (DR) by mediating angiogenesis.

Phenolic profile and antioxidant activity of Euonymus japonicus Thunb.

The aim of this study was to characterise the phenolic profile and evaluate the antioxidant activity of aqueous and methanol leaf extracts from Euonymus japonicus Thunb., a species of flowering plant in the Celastraceae family. Liquid chromatography (HPLC) with diode array and electrospray-tandem mass spectrometry (DAD-ESI-MSn) detection was used to characterise and quantify phenolic compounds. Thirty-two compounds were detected; among them, flavonoids (mainly quercetin and kaempferol glycosides) were the most abundant compounds in both extracts. The results obtained for DPPH and ABTS assays in the extracts were in agreement with the highest content of phenolics in the methanol extract.

Effects of arbuscular mycorrhizal inoculation on the growth, photosynthesis and antioxidant enzymatic activity of Euonymus maackii Rupr. under gradient water deficit levels.

The role of arbuscular mycorrhizal (AM) fungus (Rhizophagus intraradices) in the amelioration of the water deficit-mediated negative influence on the growth, photosynthesis, and antioxidant system in Euonymus maackii Rupr. was examined. E. maackii seedlings were subjected to 5 water deficit levels, soil water contents of 20%, 40%, 60%, 80% and 100% field capacity (FC), and 2 inoculation treatments, with and without AM inoculation. The water deficit increasingly limited the seedling height, biomass accumulation in shoots and roots, chlorophyll content, gas exchange and chlorophyll fluorescence parameters with an increasing water deficit level. In addition, water deficit stimulated the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), in both shoots and roots, except under 20% FC conditions. E. maackii seedlings under all water deficit conditions formed symbiosis well with AM fungi, which significantly ameliorated the drought-mediated negative effect, especially under 40% and 60% FC conditions. Under 40% to 80% FC conditions, AM formation improved seedling growth and photosynthesis by significantly enhancing the biomass accumulation, chlorophyll content and assimilation. Mycorrhizal seedlings showed better tolerance and less sensitivity to a water deficit, reflected in the lower SOD activities of shoots and roots and CAT activity of shoots under 40% and 60% FC conditions. Downregulation of the antioxidant system in mycorrhizal seedlings suggested better maintenance of redox homeostasis and protection of metabolism, including biomass accumulation and assimilation. All the results advocated the positive role of R. intraradices inoculation in E. maackii against a water deficit, especially under 40% FC, which suggested the distinct AM performance in drought tolerance and the potential role of the combination of E. maackii-AM fungi in ecological restoration in arid regions.

Isolation and purification of flavonoids from Euonymus alatus by high-speed countercurrent chromatography and neuroprotective effect of rhamnazin-3-O-rutinoside in vitro.

The flavonoids from Euonymus alatus exhibit many biological activities including significant antioxidant, anti-inflammatory, anti-cancer. In this work, a high-speed countercurrent chromatography method for the isolation and purification of flavonoids from crude extracts of Euonymus alatus was established. The effects of several solvent systems on the separation efficiency of target compounds in the extract of Euonymus alatus were studied. The solvent system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of (3:5:3:5, v/v) was chosen, in which the lower phase was used as the mobile phase at the rotation speed of 800 rpm and flow rate of 2.0 mL/min. The three flavonoids were obtained and identified as patuletin-3-O-rutinoside, rhamnazin-3-O-rutinoside, and dehydrodicatechin A by mass spectroscopy and nuclear magnetic resonance, and the quantities of patuletin-3-O-rutinoside, rhamnazin-3-O-rutinoside, and dehydrodicatechin A were 2.2, 9.7, and 1.8 mg, respectively. The results indicated that high-speed countercurrent chromatography was a simple and efficient method for the isolation and purification of flavonoids from the crude extracts of Euonymus alatus. The cellular antioxidant activity experimental result indicated that rhamnazin-3-O-rutinoside could alleviate H2 O2 -induced oxidative stress.

Intraspecific differences in plant functional traits are related to urban atmospheric particulate matter.

BACKGROUND: Functional trait-based ecological research has been instrumental in advancing our understanding of environmental changes. It is still, however, unclear how the functional traits of urban plants respond to atmospheric particulate matter, and which trade-off strategies are shown. In order to explore the variation of plant functional traits with the gradient of urban atmospheric particulate matter, we divided atmospheric particulate matter into three levels according to road distance, and measured the variation of six essential leaf functional traits and their trade-off strategies. RESULTS: Here, we show that the functional traits of plants can be used as predictors of plant response to urban atmospheric particulate matter. Within the study, leaf thickness, leaf dry matter content, leaf tissue density, stomatal density were positively correlated with atmospheric particulate matter. On the contrary, chlorophyll content index and specific leaf area were negatively correlated with atmospheric particulate matter. Plants can improve the efficiency of gas exchange by optimizing the spatial distribution of leaf stomata. Under the atmospheric particulate matter environment, urban plants show a trade-off relationship of economics spectrum traits at the intraspecific level. CONCLUSION: Under the influence of urban atmospheric particulate matter, urban plant shows a "slow investment-return" type in the leaf economics spectrum at the intraspecific level, with lower specific leaf area, lower chlorophyll content index, ticker leaves, higher leaf dry matter content, higher leaf tissue density and higher stomatal density. This finding provides a new perspective for understanding the resource trades-off strategy of plants adapting to atmospheric particulate matter.

Efficient plant regeneration from embryogenic cell suspension cultures of Euonymus alatus.

To establish an efficient plant regeneration system from cell suspension cultures of Euonymus alatus, embryogenic callus formation from immature embryos was investigated. The highest frequency of embryogenic callus formation reached 50% when the immature zygotic embryos were incubated on Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D). At higher concentrations of 2,4-D (over 2 mg/L), the frequency of embryogenic callus formation declined significantly. The total number of somatic embryos development was highest with the 3% (w/v) sucrose treatment, which was found to be the optimal concentration for somatic embryo formation. Activated charcoal (AC) and 6-benzyladenine (BA) significantly increased the frequency of plantlet conversion from somatic embryos, but gibberellic acid (GA3) had a negative effect on plantlet conversion and subsequent development from somatic embryos. Even though the cell suspension cultures were maintained for more than 1 year, cell aggregates from embryogenic cell suspension cultures were successfully converted into normal somatic embryos with two cotyledons. To our knowledge, this is the first successful report of a plant regeneration system of E. alatus via somatic embryogenesis. Thus, the embryogenic cell line and plant regeneration system established in this study can be applied to mass proliferation and production of pharmaceutical metabolite in E. alatus.

Dynamics of particle retention and physiology in Euonymus japonicus Thunb. var. aurea-marginatus Hort. with severe exhaust exposure under continuous drought.

Frequent drought events and particulate matter pollution from vehicular exhaust seriously affect urban plant growth and provisioning of ecological services. Yet, how plants respond physiologically and morphologically to these two combined stressors remains unknown. Here, we assessed particle retention dynamics and plant morphology and physiology of Euonymus japonicus Thunb. var. aurea-marginatus Hort. under continuous drought with severe exhaust exposure. Our results showed that continuous drought insignificantly lowered particle retention in each of three size fractions by 1.02 µg·cm-2 on average in the first 28 days, but significantly lowered total particle retention by 35.75 µg·cm-2 on the 35th day. We observed evident changes in morphology, leaf mass per area (LMA), pigments, gas exchange in all stressed plants. Compared with single stress, combined drought and pollution caused earlier yellowing and shedding of old leaves, significantly lowered LMA by 1.21 mg·cm-2, caused a greater decline in pigments and net photosynthetic rate (Pn). Large particles may mainly explain pigment reduction, lower weekly LMA increases, and stomatal restriction, while coarse particles may be the main drivers of the decline in Pn. Continuous drought mediated the influence of all three particle sizes on some parameters, such as weakening the impact of total particles on LMA, strengthening the impact of fine particles on photosynthesis. Our findings suggest that drought accelerates the physiological responses of plants to exhaust pollution. Under controlled severe exhaust pollution conditions, the optimal time to maintain high particle retention during continuous drought without decline in physiological conditions for E. japonicus var. aurea-marginatus was 14 days. Some additional interventions after 14 days (it could be postponed appropriately under field conditions) may help ensure healthy growth of plants and retention of particulate matter.

α-Glucosidase inhibitory triterpenoids from Euonymus fortunei.

α-Glucosidase plays an important role in catalyzing the hydrolytic cleavage of disaccharides into monosaccharides. In this study, a phytochemical investigation of the potential α-glucosidase inhibitory fraction from the aerial parts of Euonymus fortunei led to the isolation and identification of two new tetracyclic triterpenoids, fortunenones A and B (1-2), together with 11 known triterpenoids (3-13). Fortunenones A and B are rare C32 triterpenoids possessing a 24,24-dimethyl group. The partial isolated compounds were evaluated their effects on α-glucosidase, of which echinochlorin D (5), lupenone (7), wilforlide B (12), and wilforlide A (13) exhibited remarkable inhibitory effects with the half inhibitory concentration ranged from 207.2 × 10-6 M to 388.3 × 10-6 M compared with the positive control, acarbose. An enzyme kinetics analysis by Lineweaver-Burk plots revealed that the inhibition types of the four active compounds were all mixed inhibition. Molecular docking further revealed that hydrophobic interactions and hydrogen bonds play an important role in the inhibition of α-glucosidase activity. Our results demonstrate the potential of E. fortunei extract and its constituents to inhibit α-glucosidase.