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ELO-like elongase is a condensing enzyme elongating long chain fatty acids in eukaryotes. Eranthis hyemalis ELO-like elongase (EhELO1) is the first higher plant ELO-type elongase that is highly active in elongating a wide range of polyunsaturated fatty acids (PUFAs) and some monounsaturated fatty acids (MUFAs). This study attempted using domain swapping and site-directed mutagenesis of EhELO1 and EhELO2, a close homologue of EhELO1 but with no apparent elongase activity, to elucidate the structural determinants critical for catalytic activity and substrate specificity. Domain swapping analysis of the two showed that subdomain B in the C-terminal half of EhELO1 is essential for MUFA elongation while subdomain C in the C-terminal half of EhELO1 is essential for both PUFA and MUFA elongations, implying these regions are critical in defining the architecture of the substrate tunnel for substrate specificity. Site-directed mutagenesis showed that the glycine at position 220 in the subdomain C plays a key role in differentiating the function of the two elongases. In addition, valine at 161 and cysteine at 165 in subdomain A also play critical roles in defining the architecture of the deep substrate tunnel, thereby contributing significantly to the acceptance of, and interaction with primer substrates.

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This review summarizes information about the chemical composition and beneficial properties of species of the genus Eranthis Salisb. from the world's flora. To date, seven out of ~14 species found in Asia and parts of Europe have been studied to various degrees. Here, data are presented on the diversity of sets of chromones, furochromones, triterpene saponins, coumarins, and other classes of secondary metabolites of Eranthis species according to the literature. For new compounds-isolated from Eranthis for the first time-structural formulas are also provided. Among the new compounds, chromones and coumarins predominate, as do triterpene saponins of the olean and cycloartane series and lectin. The results of pharmacological studies are presented showing anti-inflammatory, antioxidant, antiviral, and other types of biological activities found in extracts, in their fractions, and in individual compounds of the aboveground and underground organs and parts of Eranthis species. Despite the limited geographic range of Eranthis plants, it is possible to search for active substances, develop methods for biological and chemical synthesis of the isolated substances, and create a finished therapeutic substance based on them. In addition, it is feasible to obtain the desired standardized pure materials from Eranthis species grown in vitro.

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The Korean endemic Eranthis byunsanensis B.Y. Sun, 1993 (Ranunculaceae) is a rare plant distributed in the southwestern part of the Korean Peninsula. The complete chloroplast (cp) genome of E. byunsanensis was sequenced by next-generation sequencing (NGS) using an Illumina HiSeq X platform. The cp genome of E. byunsanensis is 160,324 bp in length with 37.9% GC content. It showed a typical quadripartite structure consisting of a pair of inverted repeats (IRs; 28,356 bp), a large single-copy region (LSC; 87,671 bp), and a small single-copy region (SSC; 15,941 bp). The cp genome comprises 130 genes including 85 protein-coding genes (PCGs), 37 tRNA genes, and eight rRNA genes. The molecular phylogenetic analysis indicates that E. byunsanensis is closely related to Eranthis stellata, both of which belong to the genus Eranthis.

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A comparative karyotype analysis of four species of yellow-flowered Eranthis sect. Eranthis, i.e., E. bulgarica, E. cilicica, E. hyemalis, and E. longistipitata from different areas, has been carried out for the first time. All the studied specimens had somatic chromosome number 2n = 16 with basic chromosome number x = 8. Karyotypes of the investigated plants included five pairs of metacentric chromosomes and three pairs of submetacentric/subtelocentric chromosomes. The chromosome sets of the investigated species differ mainly in the ratio of submetacentric/subtelocentric chromosomes, their relative lengths, and arm ratios. A new oligonucleotide probe was developed and tested to detect 45S rDNA clusters. Using this probe and an oligonucleotide probe to 5S rDNA, 45S and 5S rDNA clusters were localized for the first time on chromosomes of E. cilicica, E. hyemalis, and E. longistipitata. Major 45S rDNA clusters were identified on satellite chromosomes in all the species; in E. cilicica, minor clusters were also identified in the terminal regions of one metacentric chromosome pair. The number and distribution of 5S rDNA clusters is more specific. In E. cilicica, two major clusters were identified in the pericentromeric region of a pair of metacentric chromosomes. Two major clusters in the pericentromeric region of a pair of submetacentric chromosomes and two major clusters in the interstitial region of a pair of metacentric chromosomes were observed in E. longistipitata. E. hyemalis has many clusters of different sizes, localized mainly in the pericentromeric regions. Summarizing new data on the karyotype structure of E. sect. Eranthis and previously obtained data on E. sect. Shibateranthis allowed conclusions to be formed about the clear interspecific karyological differences of the genus Eranthis.

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Genetic differentiation between populations is determined by various factors, including gene flow, selection, mutation, and genetic drift. Among these, gene flow is known to counter genetic differentiation. The genus Eranthis, an early flowering perennial herb, can serve as a good model to study genetic differentiation and gene flow due to its easily detectable population characteristics and known reproductive strategies, which can be associated with gene flow patterns. Eranthis populations are typically small and geographically separated from the others. Moreover, previous studies and our own observations suggest that seed and pollen dispersal between Eranthis populations is highly unlikely and therefore, currently, gene flow may not be probable in this genus. Based on these premises, we hypothesized that the genetic differentiation between the Eranthis populations would be significant, and that the genetic differentiation would not sensitively reflect geographic distance in the absence of gene flow. To test these hypotheses, genetic differentiation, genetic distance, isolation by distance, historical gene flow, and bottlenecks were analyzed in four species of this genus. Genetic differentiation was significantly high, and in many cases, extremely high. Moreover, genetic differentiation and geographic distance were positively correlated in most cases. We provide possible explanations for these observations. First, we suggest that the combination of the marker type used in our study (chloroplast microsatellites), genetic drift, and possibly selection might have resulted in the extremely high genetic differentiation observed herein. Additionally, we provide the possibility that genetic distance reflects geographic distance through historical gene flow, or adaptation in the absence of historical gene flow. Nevertheless, our explanations can be more rigorously examined and further refined through additional observations and various population genetic analyses. In particular, we suggest that other accessible populations of the genus Eranthis should be included in future studies to better characterize the intriguing population dynamics of this genus.

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Flowers are an innovative characteristic of angiosperms, and elaborate petals usually have highly specialized structures to adapt to different living environments and pollinators. Petals of Eranthis have complex bilabiate structures with nectaries and pseudonectaries; however, the diversity of the petal micromorphology and structure is unknown. Petal development, micromorphology, structure and ultrastructure in four Eranthis species were investigated under SEM, TEM and LM. The results show that petals undergo 5 developmental stages, and accessory structure formation (stage 4) mainly determines the diversity of final mature petal morphology and pseudonectaries; the central depression formed in stage 2 will develop into nectary tissues. Petals are bilabiate and have hidden nectaries in nectary grooves; they consist of one layer of rounded and raised secretory epidermal cells and 3-14 layers of secretory cells with abundant plasmodesmata between cells. A large number of sieve tubes are distributed between the cells and extend to the epidermis; in addition, the vessel elements are located below the secretory area. Nectar is stored in the intercellular space between secretory parenchyma cells and escapes through microchannels or cell rupture. Pseudonectaries in all species of Eranthis except for E. hyemalis consist of smooth, ornamented epidermal cells and 9-12 layers of parenchyma cells with sparse cytoplasm, which may have the function of attracting pollinators.

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Eranthis longistipitata Regel is an endemic plant of Central Asia. The flavonoid profile of E. longistipitata leaves was studied by mass spectrometry for the first time (natural populations of Kyrgyzstan and Uzbekistan, in 70% aqueous-ethanol extracts by liquid chromatography coupled with high-resolution mass spectrometry). Mass spectrometry revealed 18 flavonoid compounds. Flavonols featured the highest diversity, and 10 such substances were identified: 2 free aglycones (quercetin and kaempferol), 6 quercetin glycosides (peltatoside, hyperoside, reynoutrin, quercetin 3-sambubioside, rutin, and isoquercitrin), and 2 kaempferol glycosides (juglalin and trifolin). Two flavans (cianidanol and auriculoside), two hydroxyflavanones (6-methoxytaxifolin and aromadendrin), and one C-glycoside flavone-carlinoside-were identified. Dihydroxychalcones aspalathin, phloridzin, and phloretin were found too. Levels of rutin, quercetin, kaempferol, and hyperoside were confirmed by means of standards and high-performance liquid chromatography. Rutin concentration was the highest among all other identified flavonoid compounds: in the leaf samples from Kyrgyzstan, it ranged from 2.46 to 3.20 mg/g, and in those from Uzbekistan, from 1.50 to 3.01 mg/g. The diversity of flavonoid compounds in E. longistipitata leaves is probably due to external ecological and geographic factors and adaptive mechanisms.

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Docosadienoic acid (DDA, 22:2-13,16) and docosatrienoic acid (DTA, 22:3-13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in the effective incorporation of DDA and DTA at the sn-2 position of TAGs, thereby increasing the total amount of DDA and DTA in transgenic seeds. To our knowledge, this is the first plant LPAAT that can incorporate VLCPUFAs into TAGs. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.

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Aqueous-ethanol extracts (70%) from the leaves of Eranthis longistipitata Regel. (Ranunculaceae Juss.)-collected from natural populations of Kyrgyzstan-were studied by liquid chromatography with high-resolution mass spectrometry (LC-HRMS). There was no variation of the metabolic profiles among plants that were collected from different populations. More than 160 compounds were found in the leaves, of which 72 were identified to the class level and 58 to the individual-compound level. The class of flavonoids proved to be the most widely represented (19 compounds), including six aglycones [quercetin, kaempferol, aromadendrin, 6-methoxytaxifolin, phloretin, and (+)-catechin] and mono- and diglycosides (the other 13 compounds). In the analyzed samples of E. longistipitata, 14 fatty acid-related compounds were identified, but coumarins and furochromones that were found in E. longistipitata were the most interesting result; furochromones khelloside, khellin, visnagin, and cimifugin were found in E. longistipitata for the first time. Coumarins 5,7-dihydroxy-4-methylcoumarin, scoparone, fraxetin, and luvangetin and furochromones methoxsalen, 5-O-methylvisammioside, and visamminol-3'-O-glucoside were detected for the first time in the genus Eranthis Salisb. For all the above compounds, the structural formulas are given. Furthermore, detailed information (with structural formulas) is provided on the diversity of chromones and furochromones in other representatives of Eranthis. The presence of chromones in plants of the genus Eranthis confirms its closeness to the genus Actaea L. because chromones are synthesized by normal physiological processes only in these members of the Ranunculaceae family.

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Comparative karyomorphological analyses of six out of the eight white-flowered species of Eranthissect.Shibateranthis have been carried out. All studied specimens of E.byunsanensis, E.lobulata, E.pinnatifida, and E.stellata had a somatic chromosome number 2n = 16 with basic chromosome number x = 8. On the contrary, E.tanhoensis and E.sibirica had a basic chromosome number x = 7. The specimens of E.tanhoensis were diploid with 2n = 14, while the specimens of E.sibirica were polyploid with 2n = 42. Monoploid chromosome sets of the investigated diploid species had 4-5 metacentric chromosomes and 2-4 submetacentric/subtelocentric/acrocentric chromosomes. The highest level of interchromosomal asymmetry, estimated via CVCL, was found in E.byunsanensis and E.pinnatifida. The highest levels of intrachromosomal asymmetry (MCA) and heterogeneity in centromere position (CVCI) were found in E.lobulata and E.byunsanensis, while E.sibirica had the most symmetric karyotype. A multivariate PCoA analysis of basic karyotype parameters (2n, x, THL, CVCL, MCA, and CVCI) highlighted no overlap among species accessions, which was also confirmed by LDA. The average absolute monoploid DNA content (1Cx) of the 23 investigated samples of six Eranthis species varied from 9.26 ± 0.25 pg in E.sibirica to 15.93 ± 0.32 pg in E.stellata. Overall karyological affinity was highlighted between E.lobulata and E.stellata, on one side, and between E.byunsanensis and E.pinnatifida, on the other side. Interestingly, there was no significant correlation between total haploid (monoploid) chromosome length (THL) and 1Cx values in these species.

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The temporal and spatial origins and evolution of the genus Eranthis have not been previously studied. We investigated the speciation and establishment histories of four Eranthis species: Eranthis byunsanensis, E. pungdoensis, E. stellata, and E. pinnatifida. The sampling localities were Korea, Japan, Jilin in China, and the area near Vladivostok in Primorskiy, Russia. We used 12 chloroplast microsatellite loci (n = 935 individuals) and two chloroplast noncoding regions (rpl16 intron, petL-psbE intergenic spacer; n = 33 individuals). The genetic diversity, genetic structure, phylogenetic relationships of the four species were analyzed, and their ancestral areas were reconstructed. The high genetic diversity of the Jeju island population of E. byunsanensis and Russian populations of E. stellata indicated these species' northward and southward dispersal, respectively. The genetic structure analyses suggest that the populations in these four species have limited geographical structure, except for the Chinese E. stellata population (SCP). The phylogenetic analyses suggest that E. byunsanensis and E. pinnatifida are sister species and that Chinese SCP may not belong to E. stellata. The ancestral area reconstruction revealed that the most recent common ancestor of the four species existed in the current Chinese habitat of E. stellata. This study shows that E. byunsanensis and E. pinnatifida originated from a southern Eranthis species and speciated into their current forms near Jeju island and near western regions of Japan, respectively, during the Miocene. E. stellata may have dispersed southward on and near the Korean peninsula, though its specific origin remains unclear. Interestingly, the Chinese E. stellata population SCP suggests that the Chinese population might be most ancient among all the four Eranthis species. E. pungdoensis may have allopatrically speciated from E. byunsanensis during the Holocene. The Korean peninsula and the surrounding areas can be considered interesting regions which provide the opportunity to observe both northern- and southern-sourced Eranthis species.

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Eranthis stellate belong to Ranunculaceae, which is interest in phylogenetic research because it has often been considered one of the most basal eudicots families. However, there are few chloroplastg enome data of Ranunculaceae available. Here, to provide available genomic data for the phylogenetic of Ranunculaceae, we determined the complete chloroplast genome of E. stellate. The complete chloroplast sequence is 158,817 bp, including a large single-copy (LSC) region of 87,137 bp, a small single-copy (SSC) region of 16,834 bp, a pair of invert repeats (IR) regions of 27,424 bp. Plastid genome contain129 genes, 84 protein-coding genes, 36 tRNA genes, and 8 rRNA genes. Phylogenetic analysis based on 14 chloroplast genomes indicates that E. stellata is sister to Aconitum austrokoreense clade in Ranunculaceae.

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Phytochemical analysis of the tubers of Eranthis cilicica was performed as part of our continuous study on the plants of the family Ranunculaceae, which resulted in the isolation of eleven new cycloartane glycosides (1⁻11) and one new oleanane glycoside (13), together with one known oleanane glycoside (12). The structures of the new compounds were determined by extensive spectroscopic analysis, including two-dimensional (2D) NMR, and enzymatic hydrolysis followed by either X-ray crystallographic or chromatographic analysis. The aglycone (1a) of 2 and its C-23 epimer (8a), and the oleanane glycosides (12 and 13) showed cytotoxic activity against HL-60 leukemia cells with IC50 values ranging from 10.6 µM to 101.6 µM. HL-60 cells were much more sensitive to 8a (IC50 14.8 µM) than 1a (IC50 101.1 µM), indicating that the C-23 configuration is associated with the cytotoxicity of these cycloartane derivatives. Compound 12 was revealed so as to partially induce apoptotic cell death in HL-60 cells, as was evident from morphology of HL-60 cells treated with 12.

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Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata showed that the transgenic plants produced the level of DDA and DTA at approximately 30% of the total fatty acids in seeds, and the amount of the two fatty acids remained stable over four generations. The oilseed crop producing a high and sustained level of DDA and DTA provides an opportunity for high value agricultural products for nutritional and medical uses.

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Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.

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