Complete chloroplast genome and phylogenetic analysis of Anemone shikokiana.

BACKGROUND: Anemone shikokiana (Makino) Makino, disjunctly distributed in Shandong Peninsula of China and Shikoku Island of Japan, is a rare and endangered species. To provide genetic information and understand its phylogeny, we conducted research on the chloroplast (cp) genome of A. shikokiana. METHODS AND RESULTS: The complete cp genome sequence of A. shikokiana was constructed in this study. The results showed that the cp genome of A. shikokiana has a typical quadripartite cyclic with a total length of 159,286 bp. In total, 111 unique genes were identified, including 78 protein-coding genes, 29 tRNA-coding genes and 4 rRNA-coding genes. A total of 37 long repeat sequences and 67 microsatellites were found in this cp genome. The cp genome of A. shikokiana was compared with eleven other Anemone cp genomes available from the Genbank database. We found some variations among the different genomes, especially in the LSC and SSC regions, and identified some regions as potential molecular markers such as ycf1, ndhE, ndhD, ndhF-trnL, ndhA and ndhF. The results of phylogenetic analysis suggested that A. narcissiflora was the closest relative of A. shikokiana. CONCLUSIONS: The results filled the gap of cp genome sequence information of A. shikokiana, laying the foundation to explore the evolutionary relationships of A. shikokiana in future studies. It provided a valuable genetic resource for the molecular identification and phylogenetic study of Anemone.

Appropriate Reference Genes for RT-qPCR Normalization in Various Organs of Anemone flaccida Fr. Schmidt at Different Growing Stages.

Anemone flaccida Fr. Schmidt is a traditional medicinal herb in southwestern China and has multiple pharmacological effects on bruise injuries and rheumatoid arthritis (RA). A new drug with a good curative effect on RA has recently been developed from the extract of A. flaccida rhizomes, of which the main medicinal ingredients are triterpenoid saponins. Due to excessive exploitation, the wild population has been scarce and endangered in a few of its natural habitats and research on the cultivation of the plant commenced. Studies on the gene expressions related to the biosynthesis of triterpenoid saponins are not only helpful for understanding the effects of environmental factors on the medicinal ingredient accumulations but also necessary for monitoring the herb quality of the cultivated plants. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) as a sensitive and powerful technique has been widely used to detect gene expression across tissues in plants at different stages; however, its accuracy and reliability depend largely on the reference gene selection. In this study, the expressions of 10 candidate reference genes were evaluated in various organs of the wild and cultivated plants at different stages, using the algorithms of geNorm, NormFinder and BestKeeper, respectively. The purpose of this study was to identify the suitable reference genes for RT-qPCR detection in A. flaccida. The results showed that two reference genes were sufficient for RT-qPCR data normalization in A. flaccida. PUBQ and ETIF1a can be used as suitable reference genes in most organs at various stages because of their expression stabilitywhereas the PUBQ and EF1Α genes were desirable in the rhizomes of the plant at the vegetative stage.

The Double-Layer Method to the Genesis of Androgenic Plants in Anemone coronaria.

Homozygous lines occur for plant breeding programs and for studies about gene expression and genetic mapping and they can be derived from anther culture. In this chapter, the method to obtain androgenic plants from an ornamental cut flower, Anemone coronaria belonging to the Ranunculaceae family, is described. In this species, androgenic plants were obtained culturing anthers with responsive microspores in Petri dishes containing a double layer of substrate with specific composition. Moreover, thermic treatment has been applied to induce the switch from pollen development program to embryo development program. The method allows to produce both double-haploid plants from diploid mothers (2n) and di-haploid plants from tetraploid mothers (4n).

A two-step approach for systematic identification and quality evaluation of wild and introduced Anemone flaccida Fr. Schmidt (Di Wu) based on DNA barcode and UPLC-QTOF-MS/MS.

Herbal materials have both medicinal and commercial values. As such, accurate species and content identification and verification are necessary to ensure the safe and effective use for medical and commodity purposes. Herein, we introduce a two-step approach for systematic identification and quality evaluation of wild and introduced Anemone flaccida Fr. Schmidt (aka Di Wu) using DNA barcode and ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS). To begin, a precise and rapid identification method based on internal transcribed spacer 2 (ITS2) sequence was developed to ensure the authenticity of 'Di Wu' species. Next, the major active components were fully characterized utilizing a targeted profile of oleanane-type triterpenoid saponins, which was established via UPLC-QTOF-MS/MS. As a result, 34 oleanane-type triterpenoid saponins were identified or characterized in 'Di Wu.' The qualitative and relative quantitative analysis showed obvious differences between wild and introduced 'Di Wu.' Furthermore, dynamic changes in the contents of triterpenoid saponins throughout various harvesting periods were clearly explained and mid-April was identified as the appropriate harvest time. Moreover, results indicate that the contents of five main saponins (anhuienoside E, glycosideSt-I4a, hemsgiganoside B, flaccidoside II, and hederasaponin B) are more appropriate as a quality evaluation indicator than the current quality standard. The two-step approach provides a suitable strategy to evaluate the genuine quality of wild and introduced 'Di Wu,' and can be applied to the targeted analysis of other triterpenoid saponin analogues for quality evaluation. Graphical Abstract .

Color preference and spatial distribution of glaphyrid beetles suggest a key role in the maintenance of the color polymorphism in the peacock anemone (Anemone pavonina, Ranunculaceae) in Northern Greece.

In the Mediterranean region, a group of unrelated plant species share an unusual deep-red flower color and are pollinated by glaphyrid beetles. Some of these species possess different color morphs, but the mechanisms maintaining this color polymorphism are unknown. The peacock anemone, Anemone pavonina, is a color polymorphic species with red or purple flowers. We investigated the spatial distribution of its color morphs and its potential glaphyrid pollinators, Pygopleurus spp., along an elevational gradient on the southern slopes of Mount Olympus, Greece. We found a correlation between relative proportions of the two color morphs with both elevation and beetle abundance. At low elevations (< 1000 m a.s.l.), beetles were abundant and anemone populations comprised only red flowers. Above a steep transition zone with mixed-colored populations (c. 1000-1300 m) most flowers were purple and beetles were rare. Color-trapping experiments revealed a strong preference for red over other colors in beetles and colorimetric modeling suggests that a simple chromatic mechanism is sufficient to explain their color choices. We thus hypothesize that beetles select for red flowers and that with increasing elevation and decreasing beetle density, other flower visitors (e.g., bees) gain importance as pollinators and select for a different color.

Transcriptomic analysis in Anemone flaccida rhizomes reveals ancillary pathway for triterpene saponins biosynthesis and differential responsiveness to phytohormones.

Anemone flaccida Fr. Schmidt is a perennial medicinal herb that contains pentacyclic triterpenoid saponins as the major bioactive constituents. In China, the rhizomes are used as treatments for a variety of ailments including arthritis. However, yields of the saponins are low, and little is known about the plant's genetic background or phytohormonal responsiveness. Using one-quarter of the 454 pyrosequencing information from the Roche GS FLX Titanium platform, we performed a transcriptomic analysis to identify 157 genes putatively encoding 26 enzymes involved in the synthesis of the bioactive compounds. It was revealed that there are two biosynthetic pathways of triterpene saponins in A. flaccida. One pathway depends on ß-amyrin synthase and is similar to that found in other plants. The second, subsidiary ("backburner") pathway is catalyzed by camelliol C synthase and yields ß-amyrin as minor byproduct. Both pathways used cytochrome P450-dependent monooxygenases (CYPs) and family 1 uridine diphosphate glycosyltransferases (UGTs) to modify the triterpenoid backbone. The expression of CYPs and UGTs were quite different in roots treated with the phytohormones methyl jasmonate, salicylic acid and indole-3-acetic acid. This study provides the first large-scale transcriptional dataset for the biosynthetic pathways of triterpene saponins and their phytohormonal responsiveness in the genus Anemone.

Cloning, purification and characterization of nigrelysin, a novel actinoporin from the sea anemone Anthopleura nigrescens.

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family.

Biotic and abiotic drivers of intraspecific trait variation within plant populations of three herbaceous plant species along a latitudinal gradient.

BACKGROUND: The importance of intraspecific trait variation (ITV) is increasingly acknowledged among plant ecologists. However, our understanding of what drives ITV between individual plants (ITVBI) at the population level is still limited. Contrasting theoretical hypotheses state that ITVBI can be either suppressed (stress-reduced plasticity hypothesis) or enhanced (stress-induced variability hypothesis) under high abiotic stress. Similarly, other hypotheses predict either suppressed (niche packing hypothesis) or enhanced ITVBI (individual variation hypothesis) under high niche packing in species rich communities. In this study we assess the relative effects of both abiotic and biotic niche effects on ITVBI of four functional traits (leaf area, specific leaf area, plant height and seed mass), for three herbaceous plant species across a 2300 km long gradient in Europe. The study species were the slow colonizing Anemone nemorosa, a species with intermediate colonization rates, Milium effusum, and the fast colonizing, non-native Impatiens glandulifera. RESULTS: Climatic stress consistently increased ITVBI across species and traits. Soil nutrient stress, on the other hand, reduced ITVBI for A. nemorosa and I. glandulifera, but had a reversed effect for M. effusum. We furthermore observed a reversed effect of high niche packing on ITVBI for the fast colonizing non-native I. glandulifera (increased ITVBI), as compared to the slow colonizing native A. nemorosa and M. effusum (reduced ITVBI). Additionally, ITVBI in the fast colonizing species tended to be highest for the vegetative traits plant height and leaf area, but lowest for the measured generative trait seed mass. CONCLUSIONS: This study shows that stress can both reduce and increase ITVBI, seemingly supporting both the stress-reduced plasticity and stress-induced variability hypotheses. Similarly, niche packing effects on ITVBI supported both the niche packing hypothesis and the individual variation hypothesis. These results clearly illustrates the importance of simultaneously evaluating both abiotic and biotic factors on ITVBI. This study adds to the growing realization that within-population trait variation should not be ignored and can provide valuable ecological insights.

Phylogenetic reassessment of tribe Anemoneae (Ranunculaceae): Non-monophyly of Anemone s.l. revealed by plastid datasets.

Morphological and molecular evidence strongly supported the monophyly of tribe Anemoneae DC.; however, phylogenetic relationships among genera of this tribe have still not been fully resolved. In this study, we sampled 120 specimens representing 82 taxa of tribe Anemoneae. One nuclear ribosomal internal transcribed spacer (nrITS) and six plastid markers (atpB-rbcL, matK, psbA-trnQ, rpoB-trnC, rbcL and rps16) were amplified and sequenced. Both Maximum likelihood and Bayesian inference methods were used to reconstruct phylogenies for this tribe. Individual datasets supported all traditional genera as monophyletic, except Anemone and Clematis that were polyphyletic and paraphyletic, respectively, and revealed that the seven single-gene datasets can be split into two groups, i.e. nrITS + atpB-rbcL and the remaining five plastid markers. The combined nrITS + atpB-rbcL dataset recovered monophyly of subtribes Anemoninae (i.e. Anemone s.l.) and Clematidinae (including Anemoclema), respectively. However, the concatenated plastid dataset showed that one group of subtribes Anemoninae (Hepatica and Anemone spp. from subgenus Anemonidium) close to the clade Clematis s.l. + Anemoclema. Our results strongly supported a close relationship between Anemoclema and Clematis s.l., which included Archiclematis and Naravelia. Non-monophyly of Anemone s.l. using the plastid dataset indicates to revise as two genera, new Anemone s.l. (including Pulsatilla, Barneoudia, Oreithales and Knowltonia), Hepatica (corresponding to Anemone subgenus Anemonidium).

The impact of the Tekay chromoviral elements on genome organisation and evolution of Anemone s.l. (Ranunculaceae).

We studied the highly abundant chromoviral Tekay clade in species from three sister genera - Anemone, Pulsatilla and Hepatica (Ranunculaceae). With this clade, we performed a concomitant survey of its phylogenetic diversity, chromosomal organisation and transcriptional activity in Anemone s.l. in order to investigate dynamics of the Tekay elements at a finer scale than previously achieved in this or any other flowering clade. The phylogenetic tree built from Tekay sequences conformed to expected evolutionary relationships of the species; exceptions being A. nemorosa and A. sylvestris, which appeared more closely related that expected, and we invoke hybridisation events to explain the observed topology. The separation of elements into six clusters could be explained by episodic bursts of activity since divergence from a common ancestor at different points in their respective evolutionary histories. In Anemone s.l. the Tekay elements do not have a preferential position on chromosomes, i.e. they can have a: (i) centromeric/pericentromeric position; (ii) interstitial position in DAPI-positive AT-rich heterochromatic regions; can be (iii) dispersed throughout chromosomes; or even (iv) be absent from large heterochromatic blocks. Widespread transcriptional activity of the Tekay elements in Anemone s.l. taxa indicate that some copies of Tekay elements could still be active in this plant group, contributing to genome evolution and speciation within Anemone s.l. Identification of Tekay elements in Anemone s.l. provides valuable information for understanding how different localisation patterns might help to facilitate plant genome organisation in a structural and functional manner.

Ancient Origin of cGAS-STING Reveals Mechanism of Universal 2',3' cGAMP Signaling.

In humans, the cGAS-STING immunity pathway signals in response to cytosolic DNA via 2',3' cGAMP, a cyclic dinucleotide (CDN) second messenger containing mixed 2'-5' and 3'-5' phosphodiester bonds. Prokaryotes also produce CDNs, but these are exclusively 3' linked, and thus the evolutionary origins of human 2',3' cGAMP signaling are unknown. Here we illuminate the ancient origins of human cGAMP signaling by discovery of a functional cGAS-STING pathway in Nematostella vectensis, an anemone species >500 million years diverged from humans. Anemone cGAS appears to produce a 3',3' CDN that anemone STING recognizes through nucleobase-specific contacts not observed in human STING. Nevertheless, anemone STING binds mixed-linkage 2',3' cGAMP indistinguishably from human STING, trapping a unique structural conformation not induced by 3',3' CDNs. These results reveal that human mixed-linkage cGAMP achieves universal signaling by exploiting a deeply conserved STING conformational intermediate, providing critical insight for therapeutic targeting of the STING pathway.

The effect on the transcriptome of Anemone coronaria following infection with rust (Tranzschelia discolor).

In order to understand plant/pathogen interaction, the transcriptome of uninfected (1S) and infected (2I) plant was sequenced at 3'end by the GS FLX 454 platform. De novo assembly of high-quality reads generated 27,231 contigs leaving 37,191 singletons in the 1S and 38,393 in the 2I libraries. ESTcalc tool suggested that 71% of the transcriptome had been captured, with 99% of the genes present being represented by at least one read. Unigene annotation showed that 50.5% of the predicted translation products shared significant homology with protein sequences in GenBank. In all 253 differential transcript abundance (DTAs) were in higher abundance and 52 in lower abundance in the 2I library. 128 higher abundance DTA genes were of fungal origin and 49 were clearly plant sequences. A tBLASTn-based search of the sequences using as query the full length predicted polypeptide product of 50 R genes identified 16 R gene products. Only one R gene (PGIP) was up-regulated. The response of the plant to fungal invasion included the up-regulation of several pathogenesis related protein (PR) genes involved in JA signaling and other genes associated with defense response and down regulation of cell wall associated genes, non-race-specific disease resistance1 (NDR1) and other genes like myb, presqualene diphosphate phosphatase (PSDPase), a UDP-glycosyltransferase 74E2-like (UGT). The DTA genes identified here should provide a basis for understanding the A. coronaria/T. discolor interaction and leads for biotechnology-based disease resistance breeding.

Bright fluorescence monitoring system utilizing Zoanthus sp. green fluorescent protein (ZsGreen) for human G-protein-coupled receptor signaling in microbial yeast cells.

G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer's disease and Parkinson's disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).

Natural selection and neutral evolution jointly drive population divergence between alpine and lowland ecotypes of the allopolyploid plant Anemone multifida (Ranunculaceae).

Population differentiation can be driven in large part by natural selection, but selectively neutral evolution can play a prominent role in shaping patters of population divergence. The decomposition of the evolutionary history of populations into the relative effects of natural selection and selectively neutral evolution enables an understanding of the causes of population divergence and adaptation. In this study, we examined heterogeneous genomic divergence between alpine and lowland ecotypes of the allopolyploid plant, Anemone multifida. Using peak height and dominant AFLP data, we quantified population differentiation at non-outlier (neutral) and outlier loci to determine the potential contribution of natural selection and selectively neutral evolution to population divergence. We found 13 candidate loci, corresponding to 2.7% of loci, with signatures of divergent natural selection between alpine and lowland populations and between alpine populations (Fst  = 0.074-0.445 at outlier loci), but neutral population differentiation was also evident between alpine populations (FST  = 0.041-0.095 at neutral loci). By examining population structure at both neutral and outlier loci, we determined that the combined effects of selection and neutral evolution are associated with the divergence of alpine populations, which may be linked to extreme abiotic conditions and isolation between alpine sites. The presence of outlier levels of genetic variation in structured populations underscores the importance of separately analyzing neutral and outlier loci to infer the relative role of divergent natural selection and neutral evolution in population divergence.

Evolutionary diversification of banded tube-dwelling anemones (Cnidaria; Ceriantharia; Isarachnanthus) in the Atlantic Ocean.

The use of molecular data for species delimitation in Anthozoa is still a very delicate issue. This is probably due to the low genetic variation found among the molecular markers (primarily mitochondrial) commonly used for Anthozoa. Ceriantharia is an anthozoan group that has not been tested for genetic divergence at the species level. Recently, all three Atlantic species described for the genus Isarachnanthus of Atlantic Ocean, were deemed synonyms based on morphological simmilarities of only one species: Isarachnanthus maderensis. Here, we aimed to verify whether genetic relationships (using COI, 16S, ITS1 and ITS2 molecular markers) confirmed morphological affinities among members of Isarachnanthus from different regions across the Atlantic Ocean. Results from four DNA markers were completely congruent and revealed that two different species exist in the Atlantic Ocean. The low identification success and substantial overlap between intra and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding, which is not true for Ceriantharia. In addition, genetic divergence within and between Ceriantharia species is more similar to that found in Medusozoa (Hydrozoa and Scyphozoa) than Anthozoa and Porifera that have divergence rates similar to typical metazoans. The two genetic species could also be separated based on micromorphological characteristics of their cnidomes. Using a specimen of Isarachnanthus bandanensis from Pacific Ocean as an outgroup, it was possible to estimate the minimum date of divergence between the clades. The cladogenesis event that formed the species of the Atlantic Ocean is estimated to have occured around 8.5 million years ago (Miocene) and several possible speciation scenarios are discussed.

Evolution of the tetraploid Anemone multifida (2n = 32) and hexaploid A. baldensis (2n = 48) (Ranunculaceae) was accompanied by rDNA loci loss and intergenomic translocation: evidence for their common genome origin.

BACKGROUND AND AIMS: In the genus Anemone two small groups of taxa occur with the highest ploidy levels 2n = 6x = 48, belonging to the closely related clades: the montane/alpine Baldensis clade and the more temperate Multifida clade. To understand the formation of polyploids within these groups, the evolution of allohexaploid A. baldensis (AABBDD, 2n = 6x = 48) from Europe and allotetraploid Anemone multifida (BBDD, 2n = 4x = 32) from America was analysed. METHODS: Internal transcribed spacer and non-transcribed spacer sequences were used as molecular markers for phylogenetic analyses. Cytogenetic studies, including genomic in situ hybridization with genomic DNA of potential parental species as probe, fluorescence in situ hybridization with 5S and 18S rDNA as probes and 18S rDNA restriction analyses, were used to identify the parental origin of chromosomes and to study genomic changes following polyploidization. KEY RESULTS: This study shows that A. multifida (BBDD, 2n= 4x = 32) and A. baldensis (AABBDD, 2n = 6x = 48) are allopolyploids originating from the crosses of diploid members of the Multifida (donor of the A and B subgenomes) and Baldensis groups (donor of the D subgenome). The A and B subgenomes are closely related to the genomes of A. sylvestris, A. virginiana and A. cylindrica, indicating that these species or their progeny might be the ancestral donors of the B subgenome of A. multifida and A and B subgenomes of A. baldensis. Both polyploids have undergone genomic changes such as interchromosomal translocation affecting B and D subgenomes and changes at rDNA sites. Anemone multifida has lost the 35S rDNA loci characteristic of the maternal donor (B subgenome) and maintained only the rDNA loci of the paternal donor (D subgenome). CONCLUSIONS: It is proposed that A. multifida and A. baldensis probably had a common ancestor and their evolution was facilitated by vegetation changes during the Quaternary, resulting in their present disjunctive distribution.

Development of eighteen microsatellite markers in Anemone amurensis (Ranunculaceae) and cross-amplification in congeneric species.

Polyploidy plays an important role in the evolution of plant genomes. To enable the investigation of the polyploidy events within the genus Anemone, we developed eighteen microsatellite markers from the hexaploid species A. amurensis (Ranunculaceae), and tested their transferability in five closely related species. The number of total alleles (N(A)) for each resulting locus varied from one to eight. The polymorphism information content (PIC) and Nei's genetic diversity (N(GD)) for these microsatellites ranged from 0.00 to 0.71 and 0.00 to 0.91, respectively. For each population, the N(A) was one to seven, and the values of PIC and N(GD) varied from 0.00 to 0.84 and 0.00 to 0.95, respectively. In addition, most of these microsatellites can be amplified successfully in the congeneric species. These microsatellite primers provide us an opportunity to study the polyploid evolution in the genus Anemone.

Molecular structure and chromosome distribution of three repetitive DNA families in Anemone hortensis L. (Ranunculaceae).

The structure, abundance and location of repetitive DNA sequences on chromosomes can characterize the nature of higher plant genomes. Here we report on three new repeat DNA families isolated from Anemone hortensis L.; (i) AhTR1, a family of satellite DNA (stDNA) composed of a 554-561 bp long EcoRV monomer; (ii) AhTR2, a stDNA family composed of a 743 bp long HindIII monomer and; (iii) AhDR, a repeat family composed of a 945 bp long HindIII fragment that exhibits some sequence similarity to Ty3/gypsy-like retroelements. Fluorescence in-situ hybridization (FISH) to metaphase chromosomes of A. hortensis (2n = 16) revealed that both AhTR1 and AhTR2 sequences co-localized with DAPI-positive AT-rich heterochromatic regions. AhTR1 sequences occur at intercalary DAPI bands while AhTR2 sequences occur at 8-10 terminally located heterochromatic blocks. In contrast AhDR sequences are dispersed over all chromosomes as expected of a Ty3/gypsy-like element. AhTR2 and AhTR1 repeat families include polyA- and polyT-tracks, AT/TA-motifs and a pentanucleotide sequence (CAAAA) that may have consequences for chromatin packing and sequence homogeneity. AhTR2 repeats also contain TTTAGGG motifs and degenerate variants. We suggest that they arose by interspersion of telomeric repeats with subtelomeric repeats, before hybrid unit(s) amplified through the heterochromatic domain. The three repetitive DNA families together occupy approximately 10% of the A. hortensis genome. Comparative analyses of eight Anemone species revealed that the divergence of the A. hortensis genome was accompanied by considerable modification and/or amplification of repeats.

Cloning and characterization of unusual fatty acid desaturases from Anemone leveillei: identification of an acyl-coenzyme A C20 Delta5-desaturase responsible for the synthesis of sciadonic acid.

The seed oil of Anemone leveillei contains significant amounts of sciadonic acid (20:3Delta(5,11,14); SA), an unusual non-methylene-interrupted fatty acid with pharmaceutical potential similar to arachidonic acid. Two candidate cDNAs (AL10 and AL21) for the C(20) Delta(5cis)-desaturase from developing seeds of A. leveillei were functionally characterized in transgenic Arabidopsis (Arabidopsis thaliana) plants. The open reading frames of both Delta(5)-desaturases showed some similarity to presumptive acyl-coenzyme A (CoA) desaturases found in animals and plants. When expressed in transgenic Arabidopsis, AL21 showed a broad range of substrate specificity, utilizing both saturated (16:0 and 18:0) and unsaturated (18:2, n-6 and 18:3, n-3) substrates. In contrast, AL10 did not show any activity in wild-type Arabidopsis. Coexpression of AL10 or AL21 with a C(18) Delta(9)-elongase in transgenic Arabidopsis plants resulted in the production of SA and juniperonic fatty acid (20:4Delta(5,11,14,17)). Thus, AL10 acted only on C(20) polyunsaturated fatty acids in a manner analogous to "front-end" desaturases. However, neither AL10 nor AL21 contain the cytochrome b(5) domain normally present in this class of enzymes. Acyl-CoA profiling of transgenic Arabidopsis plants and developing A. leveillei seeds revealed significant accumulation of Delta(5)-unsaturated fatty acids as acyl-CoAs compared to the accumulation of these fatty acids in total lipids. Positional analysis of triacylglycerols of A. leveillei seeds showed that Delta(5)-desaturated fatty acids were present in both sn-2 and sn-1 + sn-3 positions, although the majority of 16:1Delta(5), 18:1Delta(5), and SA was present at the sn-2 position. Our data provide biochemical evidence for the A. leveillei Delta(5)-desaturases using acyl-CoA substrates.