Antibacterial Effect of Euryale ferox Seed Shell Polyphenol Extract on Salmonella Typhimurium.

This study aimed to evaluate the effects of Euryale ferox Seed Shell Polyphenol Extract (EFSSPE) on a foodborne pathogenic bacterium. EFSSPE showed antimicrobial activity toward Salmonella Typhimurium CICC 22956; the minimum inhibitory concentration of EFSSPE was 1.25 mg/mL, the inhibition curve also reflected the inhibitory effect of EFSSPE on the growth of S. Typhimurium. Detection of alkaline phosphatase outside the cell revealed that EFSSPE treatment damaged the cell wall integrity of S. Typhimurium. EFSSPE also altered the membrane integrity, thereby causing leaching of 260-nm-absorbing material (bacterial proteins and DNA). Moreover, the activities of succinate dehydrogenase and malate dehydrogenase were inhibited by EFSSPE. The hydrophobicity and clustering ability of cells were affected by EFSSPE. Scanning electron microscopy showed that EFSSPE treatment damaged the morphology of the tested bacteria. These results indicate that EFSSPE can destroy the cell wall integrity and alter the permeability of the cell membrane of S. Typhimurium.

Potential application of nanoliposomes loaded with complex tannins from the seed shell of Euryale ferox in the anti-browning of fresh-cut asparagus lettuce.

Surface browning of plant-derived fresh-cut products is mainly caused by conversion of the phenolic compounds into o-quinones under tyrosinase catalysis. In this study, the rarely reported complex tannins from Euryale ferox seed shell (ECTs) constituted by the units of 35.60% condensed tannins and 64.40% hydrolysable tannins were shown to suppress the activity of tyrosinase efficiently, supporting the exploitation of ECTs into novel anti-browning agents. However, the utilization of ECTs in food preservation is often restricted because of their chemical instability to external environment. Further fabrication of nanoliposomes loaded with ECTs (ECTs-NLs) herein was carried out to improve the stability of ECTs. DLS, TEM, FTIR, DSC and XRD confirmed that ECTs were encapsulated into nanoliposomes successfully, and ECTs-NLs appeared as vesicle-like spherical morphology with favorable encapsulation efficiency, uniform particle size distribution and negative zeta-potential. The resulting ECTs-NLs were relatively stable in the dark at 4 °C. Nanoliposomal encapsulation significantly enhanced ECTs stability, thus protecting inhibitory effect of ECTs against tyrosinase. Furthermore, anti-browning evaluation proved that ECTs-NLs had distinct advantages over free ECTs in alleviating surface browning of fresh-cut asparagus lettuces. These results suggested that nanoliposomes were effective in stabilizing ECTs and ECTs-NLs could be potentially applied to the fresh-cut food industry.

[Antioxidant activity of Euryale ferox seed shell extract and its therapeutic effects on oral ulcer in rats].

OBJECTIVE: To investigate the therapeutic effect of Euryale ferox seed shell extract on oral ulcer in rats and its underlying mechanism. METHODS: The contents of polyphenols and flavonoids in Euryale ferox seed shells were determined by Folin-phenol assay and aluminum nitrate colorimetry, respectively. DPPH·, ABTS+·, ·OH and·O2- scavenging experiments were performed to evaluate the antioxidant activities of Euryale ferox seed shell extract in vitro. In a rat model of oral ulcer induced by burning with glacial acetic acid, the therapeutic effect of Euryale ferox seed shell extract was assessed by detecting changes in serum levels of oxidative factors by enzyme-linked immunosorbent assay (ELISA) and observing pathological changes of the ulcerous mucosa using HE staining; the therapeutic mechanism of the extract was explored by detecting the expression levels of Keap1, Nrf2, Nes-Nrf2 and HO-1 proteins in ulcerous mucosa using Western blotting. RESULTS: The ethyl acetate extract of Euryale ferox seed shells contained 306.74±1.04 mg/g polyphenols and 23.43±0.61 mg/g flavonoids and had IC50 values for scavenging DPPH· and ABTS+· free radicals of 3.42 ± 0.97 µg/mL and 3.32 ± 0.90 µg/mL, respectively. In the rat models, the ethyl acetate extract significantly ameliorated oral mucosal ulcer, increased serum CAT level, and decreased serum MDA level. The protein expression levels of Nes-Nrf2 and HO-1 were increased and Keap1 protein expression was lowered significantly in the ulcerous mucosa of the rats after treatment with the extract (P<0.05 or 0.01). CONCLUSION: The therapeutic effect of Euryale ferox seed shell extract on oral ulcers in rats is mediated probably by activation of the Keap1/Nrf2/HO-1 signaling pathway.

Bismuth oxymetallate-modified biochar derived from Euryale ferox husk for efficient removal of Congo red from wastewater: adsorption behavior and mechanisms.

Using Euryale ferox husk as raw material, pristine biochar (EBC), Bi2MoO6-modified biochar (BM-EBC), and BiFeO3-modified biochar (BF-EBC) were prepared and employed for decontaminating Congo red (CR) from wastewater. Compared with EBC (217.59 mg/g) and BF-EBC (359.49 mg/g), a superior adsorption capacity of 460.77 mg/g was achieved by BM-EBC. Based on the evaluation results of the Freundlich and pseudo-second-order models, multilayer chemisorption was suggested as the adsorption mechanism. The adsorption process of BM-EBC was spontaneous and endothermic, and the rate-limiting step pertained to liquid film diffusion and intraparticle diffusion. The underlying removal mechanism was explored via SEM, BET, FTIR, XPS, Raman spectra, and Zeta potential analyses. The introduction of bismuth oxymetallates with their high number of M-O (M: Bi, Mo, Fe) structural elements provided the adsorbent with enlarged surface areas and reinforced oxygen functional groups, thereby promoting pore filling, π-π interactions, hydrogen bonding, and complexation, leading to enhanced adsorption capacity. These results demonstrate that Euryale ferox husk biochar modified by bismuth oxymetallates has high prospects for valorizing biomass waste and removing CR from wastewater.

Preparation, physicochemical characterization, and in vitro starch digestibility on complex of Euryale ferox kernel starch with ferulic acid and quercetin.

The objective of the current research was to analyze the physicochemical, structural, and in vitro starch digestibility of Euryale ferox kernel starch (EFKS) in complexation with ferulic acid (FA) and quercetin (QR). XRD results have shown that FA and QR were attached to starch resulting crystalline complexes. SEM image showed a smooth, compact structure, indicating FA and QR assist in the reorganization of starch molecules. The 1H NMR spectra of starch-polyphenols complexes showed multiple additional peaks between 6.00 and 9.00 ppm due to the benzene ring and phenolic hydroxyl groups imparted from polyphenols. The shifting and emergence of the characteristic peak observed in the DSC thermogram confirmed that polyphenols were successfully attached to starch. Complexation alters colors, reduced swelling power, and increased the solubility of the complexes. Following the complexation of FA and QR, the content of resistant starch exhibited a significant rise, escalating from 7.69 % (control sample) to 49.39 % (10 % FA) and 54.68 % (10 % QR). This led to a notable reduction in the predicted glycemic index (pGI).The higher resistant starch in the complex is attributed due to the combined effects of the reordered structure of the complexes and the inhibitory activity of polyphenols against starch digestive enzymes. Therefore, EFKS-FA and EFKS-QR complex can be used as a functional ingredient for a low glycemic index food.

The Ethnopharmacological, Phytochemical, and Pharmacological Review of Euryale ferox Salisb.: A Chinese Medicine Food Homology.

Euryale ferox Salisb. (prickly water lily) is the only extent of the genus Euryale that has been widely distributed in China, India, Korea, and Japan. The seeds of E. ferox (EFS) have been categorized as superior food for 2000 years in China, based on their abundant nutrients including polysaccharides, polyphenols, sesquineolignans, tocopherols, cyclic dipeptides, glucosylsterols, cerebrosides, and triterpenoids. These constituents exert multiple pharmacological effects, such as antioxidant, hypoglycemic, cardioprotective, antibacterial, anticancer, antidepression, and hepatoprotective properties. There are very few summarized reports on E. ferox, albeit with its high nutritional value and beneficial activities. Therefore, we collected the reported literature (since 1980), medical classics, database, and pharmacopeia of E. ferox, and summarized the botanical classification, traditional uses, phytochemicals, and pharmacological effects of E. ferox, which will provide new insights for further research and development of EFS-derived functional products.

The Effect and Mechanism of Corilagin from Euryale Ferox Salisb Shell on LPS-Induced Inflammation in Raw264.7 Cells.

(1) Background: Euryale ferox Salisb is a large aquatic plant of the water lily family and an edible economic crop with medicinal value. The annual output of Euryale ferox Salisb shell in China is higher than 1000 tons, often as waste or used as fuel, resulting in waste of resources and environmental pollution. We isolated and identified the corilagin monomer from Euryale ferox Salisb shell and discovered its potential anti-inflammatory effects. This study aimed to investigate the anti-inflammatory effect of corilagin isolated from Euryale ferox Salisb shell. (2) Methods: We predict the anti-inflammatory mechanism by pharmacology. LPS was added to 264.7 cell medium to induce an inflammatory state, and the safe action range of corilagin was screened using CCK-8. The Griess method was used to determine NO content. The presence of TNF-α, IL-6, IL-1ß, and IL-10 was determined by ELISA to evaluate the effect of corilagin on the secretion of inflammatory factors, while that of reactive oxygen species was detected by flow cytometry. The gene expression levels of TNF-α, IL-6, COX-2, and iNOS were determined using qRT-PCR. qRT-PCR and Western blot were used to detect the mRNA and expression of target genes in the network pharmacologic prediction pathway. (3) Results: Network pharmacology analysis revealed that the anti-inflammatory effect of corilagin may be related to MAPK and TOLL-like receptor signaling pathways. The results demonstrated the presence of an anti-inflammatory effect, as indicated by the reduction in the level of NO, TNF-α, IL-6, IL-1ß, IL-10, and ROS in Raw264.7 cells induced by LPS. The results suggest that corilagin reduced the expression of TNF-α, IL-6, COX-2, and iNOS genes in Raw264.7 cells induced by LPS. The downregulation of the phosphorylation of IκB-α protein related to the toll-like receptor signaling pathway and upregulation of the phosphorylation of key proteins in the MAPK signaling pathway, P65 and JNK, resulted in reduced tolerance toward lipopolysaccharide, allowing for the exertion of the immune response. (4) Conclusions: The results demonstrate the significant anti-inflammatory effect of corilagin from Euryale ferox Salisb shell. This compound regulates the tolerance state of macrophages toward lipopolysaccharide through the NF-κB signaling pathway and plays an immunoregulatory role. The compound also regulates the expression of iNOS through the MAPK signaling pathway, thereby alleviating the cell damage caused by excessive NO release.

Extraction, identification, and starch-digestion inhibition of phenolics from Euryale ferox seed coat.

BACKGROUND: Euryale ferox is an important cash crop and valuable tonic in traditional medicine. The seeds of E. ferox are rich in starch, which is hard to digest, and the digestion speed is significantly slower than that of rice starch. The goal of this study was to evaluate the effects of E. ferox seed-coat phenolics (EFCPs) on the digestion of E. ferox seed starch. RESULTS: EFCPs were extracted and identified by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. We optimized the extraction parameters, and the final extraction yield was about 1.49%. We identified seven phenolics from the E. ferox seed-coat extracts: gallic acid, digalloylhexoside, catechin, procyanidin B2, epicatechin, ellagic acid, and epicatechin gallate. Quantitative analysis results showed that the E. ferox seed phenolics mainly distributed in the seed coat and the gallic acid, digalloylhexoside, and epicatechin gallate were three main phenolic compounds. The phenolics displayed strong inhibitory activities on α-glucosidase and α-amylase with an IC50 of 3.25 µg mL-1 and 1.36 mg mL-1 respectively. Furthermore, these phenolics could interact with starch by hydrogen bonds, which might make its starch more difficult to digest. CONCLUSION: Our investigation suggests that the EFCPs can strongly inhibit the digestion of E. ferox seed starch by inhibiting the α-amylase and α-glucosidase activities and interacting with starch by hydrogen bonds; therefore, E. ferox seeds have a promising application prospect in foods for hypoglycemia. © 2023 Society of Chemical Industry.

Effect of Euryale ferox seed shell extract addition on the in vitro starch digestibility and predicted glycemic index of wheat-based bread.

The inhibitory effects of Euryale ferox seed shell extract (EFSSE) on the activity of α-amylase and α-glucosidase were studied. EFSSE (0.25 % to 2 %) was used to fortify bread and analyzed the in vitro starch digestibility (IVSD) digestion kinetics, and the predicted glycemic index (pGI) was estimated. The swarm intelligence supervised neural network (SISNN) technique was applied for the predictive simulation of digestion kinetics and pGI. Principal component analysis (PCA) with proportional odds modeling (POM) was used to find the most sensitive component based on the sensory attributes of bread. The inhibitory effect of EFSSE on α-amylase and α-glucosidase in terms of IC50 was 62.95 and 52.06 µg/mL, respectively. Fortification of bread with EFSSE could affect loaf volume, hardness, and color. Euryale ferox seed shell extract could decreased the rate of hydrolysis of bread. EFSSE (2 %) had a strong inhibitory impact, as evidenced by the drop in glycemic index from 94.61 to 61.66. SISNN-based kinetics was much better as compared to mathematical modeling-based digestion kinetics. Findings of the present study have shown that EFSSE could be employed as an additive to produce lower glycemic index functional bread.

Structure and physicochemical properties of low digestible Euryale ferox Salisb. seed starch.

BACKGROUND: Euryale ferox Salisb. is widely grown in China and Southeast Asia as a grain crop and medicinal plant. The composition, morphology, structure, physicochemical properties, thermal properties, and in vitro digestibility of North Euryale ferox seeds starch (NEFS), hybrid Euryale ferox seeds starch (HEFS), and South Euryale ferox seeds starch (SEFS) were studied. RESULT: Of the varieties that were studied, the amylose content of NEFS (23.03%) was the highest. Starch granules of each variety were smooth, sharp, small, and had an average diameter of 2 µm. All three varieties were A-type crystals with crystallinity ranging from 26.42% to 28.17%. The degree of double helix and the short-range order ranged from 1.9006 to 2.5324 and 1.4294 to 1.6006, respectively. The high proportion of C1 region in NEFS (17.74%) and HEFS (17.66%) were found. Thermodynamic properties in North Euryale ferox seeds included the highest onset temperature (To ) (71.43 °C), peak temperature (Tp ) (76.60 °C), conclusion temperature (Tc ) (82.77 °C), enthalpy of gelatinization (ΔH) (12.64 J g-1 ), and peak viscosity (1514 mPa·s). All three varieties maintained a low level of in vitro digestibility, with the highest resistant starch (RS) content (29.57%), the lowest rapidly digestible starch (RDS) content (27.07%), and the slowest hydrolysis kinetic constant (0.0303) in NEFS. CONCLUSION: The results revealed that the low digestibility of NEFS was attributable to compact granules, high crystallinity, high degree of order, and strong thermal stability. These digestive, physicochemical, and thermodynamic properties provide information for the future application of Euryale ferox seed starch in the food industry. © 2022 Society of Chemical Industry.

Enhancing Bioactive Components of Euryale ferox with Lactobacillus curvatus to Reduce H2O2-Induced Oxidative Stress in Human Skin Fibroblasts.

This study investigated the effects of Lactobacillus curvatus fermentation on the oxidative stress attenuating effects of Euryale ferox on H2O2-induced human skin fibroblasts (HSF). The results showed that Lactobacillus curvatus fermentation (i) increases the content of the various bioactive components of Euryale ferox and is found to have smaller molecular weights of polysaccharides and polypeptides; (ii) increases the overall intracellular and extracellular antioxidant capacity of H2O2-induced HSF while reducing reactive oxygen species (ROS) levels. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) all showed simultaneous increases in activity. Aside from that, the Nrf2 and MAPK signaling pathways are activated to regulate downstream-associated proteins such as the Bax/Bcl-2 protein ratio, matrix metalloproteinase 1 (MMP-1) activity, and human type I collagen (COL-1). These results suggested that the fermentation of Euryale ferox with Lactobacillus curvatus enhances its antioxidant capacity and attenuates apoptosis and senescence caused by oxidative stress.

Bio-Fabrication of Euryale ferox (Makhana) Leaf Silver Nanoparticles and Their Antibacterial, Antioxidant and Cytotoxic Potential.

Bio-fabrication of green or plant extract-based silver nanoparticles has garnered much praise over the past decade as the methodology is environment-friendly, undemanding, non-pathogenic, and economical. In the current study, leaves of Eurale ferox (Makhana), considered as waste, were used for the bio-fabrication of silver nanoparticles (ELAgNPs). Various analytical techniques including UV−VIS spectroscopy, Field emission scanning electron microscopy equipped with an energy dispersive X-ray spectrometer (FESEM-EDX), Particle size analyzer (PSA), Fourier transform infra-red spectroscopy (FTIR) and high-resolution transmission electron microscopy (HRTEM) were used for their characterization. Their antibacterial efficacy was examined against gram positive bacterium, Bacillus subtilis and gram negative bacterium, Escherichia coli. The antioxidant potential of the ELAgNPs was compassed by 2, 2 diphenyl-1-picryl hydrazyl (DPPH; λmax = 517 nm) assay, H2O2 (λmax = 230 nm) and OH− (λmax = 520 nm)-based radical scavenging assays. The cytotoxicity was checked against the VERO cell line using 3-[4, 5-dimethyl thiazol-2-yl]-2, 5 diphenyl tetrazolium bromide (MTT) assay. A mean particle size of 26.51 ± 8.87 nm with a size distribution of 7.08−53.94 nm was obtained using HRTEM. The ELAgNPs exhibited dose-dependent antibacterial efficacy with a maximum zone of inhibition (ZOI) of 21.98 ± 0.59 mm against B. subtilis and of 16.46 ± 0.22 mm against E. coli at 500 ppm after 24 h of incubation. The median lethal concentration for the cytotoxicity analysis was found to be 9.54 ± 0.35 ppm, 120.9 ± 6.31 ppm, and 20.74 ± 0.63 ppm for ELAgNPs, commercial silver nanoparticles (CAgNPs), and silver nitrate (SN), respectively. The ordinary one-way ANOVA results exhibited a significant decrease in cell viability after 72 h of incubation at p < 0.05, α = 0.05. In conclusion, the ELAgNPs showed good antibacterial, radical scavenging and dose-dependent cytotoxicity against the VERO cells. Therefore, these could be used for biomedical applications. Phyto-constituents present in the plant not only act as reducing agents but also as stabilizing and coating agents, and the availability of a wide range of metabolites makes the green approach more promising.

Euryale Small Auxin Up RNA62 promotes cell elongation and seed size by altering the distribution of indole-3-acetic acid under the light.

Euryale (Euryale ferox Salisb.) is an aquatic crop used as both food and drug in Asia, but its utilization is seriously limited due to low yield. Previously, we hypothesized that Euryale small auxin up RNAs (EuSAURs) regulate seed size, but the underlying biological functions and molecular mechanisms remain unclear. Here, we observed that the hybrid Euryale lines (HL) generate larger seeds with higher indole-3-acetic acid (IAA) concentrations than those in the North Gordon Euryale (WT). Histological analysis suggested that a larger ovary in HL is attributed to longer cells around. Overexpression of EuSAUR62 in rice (Oryza sativa L.) resulted in larger glumes and grains and increased the length of glume cells. Immunofluorescence and protein interaction assays revealed that EuSAUR62 modulates IAA accumulation around the rice ovary by interacting with the rice PIN-FORMED 9, an auxin efflux carrier protein. Euryale basic region/leucine zipper 55 (EubZIP55), which was highly expressed in HL, directly binds to the EuSAUR62 promoter and activated the expression of EuSAUR62. Constant light increased the expression of both EubZIP55 and EuSAUR62 with auxin-mediated hook curvature in HL seedlings. Overall, we proposed that EuSAUR62 is a molecular bridge between light and IAA and plays a crucial role in regulating the size of the Euryale seed.

Material basis and action mechanism of Euryale Ferox Salisb in preventing and treating diabetic kidney disease.

The aim of this study was to determine the chemical structure and mechanism of action of Euryale ferox Salisb (ES) in the prevention and treatment of diabetic kidney disease (DKD). The TCMSP, SymMap V2, CTD, DisGeNET, and GeneCards databases were searched for ES components, targets, and DKD targets using the network pharmacology method to identify common drug-disease targets. PPI analysis was used to identify hub genes, which were then followed by DKD clinical relevance, GO, KEGG analysis, and transcription factor prediction. Finally, molecular docking was performed. We discovered 24 components of ES and 72 objectives of ES, 9 of which were clinically relevant and primarily regulated by transcription factors such as HNF4A and PPARG. They are involved primarily in signal transduction, inflammatory responses, TNF regulation, apoptosis, MAPK, and other signaling pathways. The main components are oleic acid targeting the protein encoded by PPARA, LPL, FABP1, and vitamin E binding the protein encoded by MAPK1, TGFB1. In general, this approach provides an effective strategy in which ES acts primarily against DKD through oleic acid and vitamin E, targeting the protein encoded by PPARA, LPL, FABP1, MAPK1 to regulate TNF, apoptosis, MAPK, and other signaling pathways. PRACTICAL APPLICATIONS: Euryale ferox Salisb (ES) is well known for its use in medicine and food. Furthermore, ES contains many nutrients, whose pharmacological properties, including antidepressant, antioxidant, and anti-diabetic action, have been extensively demonstrated by numerous studies. In this article, through network pharmacology combined with clinical correlation analysis and molecular docking, the target and mechanism of ES in the treatment of diabetic kidney disease (DKD) were discussed, which clarified its mechanism at the molecular level. Provides a reference for the further development and utilization of ES.

Effect of Thermal Treatment on the Physicochemical, Ultrastructural, and Antioxidant Characteristics of Euryale ferox Seeds and Flour.

Euryale ferox seeds (EFS) were less gelatinized, preventing the release of nutrients and functional compounds, resulting in limited applications in meals and the food industry. Nutraceutical importance of EFS includes starch, protein, lipids, 20 amino acids, minerals, and vitamins (C, E, and beta carotene). This study aimed to evaluate the effect of three different thermal treatments on EFS's physicochemical and nutritional properties and expected to improve its applicability. The results showed that the bulk density, thousand-grain weight, and hardness of thermal treated EFS were significantly decreased (p < 0.05), whereas the maximum decrease was observed in the industrial infrared heating-assisted fluidized bed (IHFH) treatment. Meanwhile, there were more crevices, fissures, and heightened porous structures in EFS between the pericarp and episperm and the endosperm after heat treatment, which facilitated grinding and water absorption. Notably, EFS's water and oil absorption capacities increased significantly (p < 0.05) with microwave and IHFH treatments. EFS ground's solubility into powder was increased significantly with thermal treatment (p < 0.05). Furthermore, the functional properties of TPC, TFC, DPPH radical scavenging activity, and reducing power were significantly increased (p < 0.05). In general, the changes in the physicochemical properties of EFS and increased bioactivity were caused by microwave and IHFH treatments. Hence, it might improve the food value of EFS while providing valuable information to researchers and food manufacturers.

EfABI4 Transcription Factor Is Involved in the Regulation of Starch Biosynthesis in Euryale ferox Salisb Seeds.

Starch is the final product of photosynthesis and the main storage form in plants. Studies have shown that there is a close synergistic regulatory relationship between ABA signal transduction and starch biosynthesis. In this study, we employed RNA sequencing (RNA-Seq) to investigate transcriptomic changes of the Euryale ferox seeds treated by exogenous ABA. The differentially expressed genes engaged in the "Starch and sucrose" and "TCA cycle" pathway. Furthermore, the key transcription factor EfABI4 in ABA signaling pathway and the key genes of starch biosynthesis (EfDBE1, EfSBE2, EfSS1, EfSS2, EfSS3, EfSS4 and EfGBSS1) were significantly up-regulated. Further, the Euryale ferox plant was treated with ABA, it was found that the total starch content of Euryale ferox seeds at different development stages was significantly higher than that of the control, and the key genes of starch synthesis in Euryale ferox seeds were also significantly up-regulated. Finally, yeast one-hybrid and dual luciferase assay proved that EfABI4 can promote the expression of EfSS1 by directly binding to its promoter. Subcellular localization results showed that EfABI4 protein was located at the nucleus and EfSS1 protein was located in the cytomembrane. These findings revealed that ABA promotes starch synthesis and accumulation by mediating EfABI4 to directly promote EfSS1 gene expression, which is helpful for understanding starch synthesis in seeds.

Proteomic analysis of Euryale ferox Salisb seeds at different developmental stages.

The development of plant seeds is accompanied by changes in their internal substances. The edible part of E. ferox is the seed, and starch and flavonoids are the storage substances and functional substances in E. ferox seeds respectively. Herein, four time points of seed development, including after flowering T10 (10 days), T20 (20 days), T30 (30 days) and T40 (40 days), were investigated by using iTRAQ technology. A total of 2809 differential proteins were identified. The enrichment analysis of differential proteins found that they were mainly enriched in starch synthesis pathways and flavonoid biosynthesis pathways. The key candidate enzymes for starch synthesis, APS (c54069), APL (c55730), SBE (c56416), SSS (c54912) and GBSS (c53181), were identified. At the same time,PAL (c50934), CHS (c49212), F3H (c35949) and ANS (c54610) may be key enzymes in flavonoid biosynthesis. In addition, the ABA signal transduction pathway was analyzed and it was identified that PYL3 (c54854) and ABI5 (c56122) are up-regulated from T10 to T40, and it is speculated that they play an important regulatory role in the development of E. ferox seeds. Together, these results reveals the dynamic changes during the development of E. ferox seeds, which will provide guidance for the study of the molecular mechanism of starch and flavonoids.

Genome-Wide Analysis of NBS-LRR Genes From an Early-Diverging Angiosperm Euryale ferox.

NBS-LRR genes are the largest gene family in plants conferring resistance to pathogens. At present, studies on the evolution of NBS-LRR genes in angiosperms mainly focused on monocots and eudicots, while studies on NBS-LRR genes in the basal angiosperms are limited. Euryale ferox represents an early-diverging angiosperm order, Nymphaeales, and confronts various pathogens during its lifetime, which can cause serious economic losses in terms of yield and quality. In this study, we performed a genome-wide identification and analysis of NBS-LRR genes in E. ferox. All 131 identified NBS-LRR genes could be divided into three subclasses according to different domain combinations, including 18 RNLs, 40 CNLs, and 73 TNLs. The E. ferox NBS-LRR genes are unevenly distributed on 29 chromosomes; 87 genes are clustered at 18 multigene loci, and 44 genes are singletons. Gene duplication analysis revealed that segmental duplications acted as a major mechanism for NBS-LRR gene expansions but not for RNL genes, because 18 RNL genes were scattered over 11 chromosomes without synteny loci, indicating that the expansion of RNL genes could have been caused by ectopic duplications. Ancestral gene reconciliation based on phylogenetic analysis revealed that there were at least 122 ancestral NBS-LRR lineages in the common ancestor of the three Nymphaeaceae species, suggesting that NBS-LRR genes expanded slightly during speciation in E. ferox. Transcriptome analysis showed that the majority of NBS-LRR genes were at a low level of expression without pathogen stimulation. Overall, this study characterized the profile of NBS-LRR genes in E. ferox and should serve as a valuable resource for disease resistance breeding in E. ferox.

The adaptive evolution of Euryale ferox to the aquatic environment through paleo-hexaploidization.

Occupation of living space is one of the main driving forces of adaptive evolution, especially for aquatic plants whose leaves float on the water surface and thus have limited living space. Euryale ferox, from the angiosperm basal family Nymphaeaceae, develops large, rapidly expanding leaves to compete for space on the water surface. Microscopic observation found that the cell proliferation of leaves is almost completed underwater, while the cell expansion occurs rapidly after they grow above water. To explore the mechanism underlying the specific development of leaves, we performed sequences assembly and analyzed the genome and transcriptome dynamics of E. ferox. Through reconstruction of the three sub-genomes generated from the paleo-hexaploidization event in E. ferox, we revealed that one sub-genome was phylogenetically closer to Victoria cruziana, which also exhibits gigantic floating leaves. Further analysis revealed that while all three sub-genomes promoted the evolution of the specific leaf development in E. ferox, the genes from the sub-genome closer to V. cruziana contributed more to this adaptive evolution. Moreover, we found that genes involved in cell proliferation and expansion, photosynthesis, and energy transportation were over-retained and showed strong expression association with the leaf development stages, such as the expression divergence of SWEET orthologs as energy uploaders and unloaders in the sink and source leaf organs of E. ferox. These findings provide novel insights into the genome evolution through polyploidization, as well as the adaptive evolution regarding the leaf development accomplished through biased gene retention and expression sub-functionalization of multi-copy genes in E. ferox.

[Expression profiling and functional verification of flavonoid 3'-hydroxylase gene from leaves of Euryale ferox].

Leaves of Euryale ferox are rich in anthocyanins. Anthocyanin synthesis is one of the important branches of the flavonoid synthesis pathway, in which flavonoid 3'-hydroxylase(F3'H) can participate in the formation of important intermediate products of anthocyanin synthesis. According to the data of E. ferox transcriptome, F3'H cDNA sequence was cloned in the leaves of E. ferox and named as EfF3'H. The correlation between EfF3'H gene expression and synthesis of flavonoids was analyzed by a series of bioinforma-tics tools and qRT-PCR. Moreover, the biological function of EfF3'H was verified by the heterologous expression in yeast. Our results showed that EfF3'H comprised a 1 566 bp open reading frame which encoded a hydrophilic transmembrane protein composed of 521 amino acid residues. It was predicted to be located in the plasma membrane. Combined with predictive analysis of conserved domains, this protein belongs to the cytochrome P450(CYP450) superfamily. The qRT-PCR results revealed that the expression level of EfF3'H was significantly different among different cultivars and was highly correlated with the content of related flavonoids in the leaves. Eukaryotic expression studies showed that EfF3'H protein had the biological activity of converting kaempferol to quercetin. In this study, EfF3'H cDNA was cloned from the leaves of E. ferox for the first time, and the biological function of the protein was verified. It provi-ded a scientific basis for further utilizing the leaves of E. ferox and laid a foundation for the further analysis of the biosynthesis pathway of flavonoids in medicinal plants.