Phytochemical and Biological Characterization of Methanolic Extracts from Rumex algeriensis and Rumex tunetanus.

The present study is aimed at the evaluation of the phytochemical profile and the biochemical properties of methanolic extracts obtained from different parts of Rumex algeriensis and Rumex tunetanus, two relict species limited to the North Africa. Phytochemical analyses of these extracts were performed using standard colorimetric procedures, HPLC-DAD, and HPLC-DAD-ESI/MS, and their antioxidant/free radical scavenging capability was estimated through several in vitro cell-free assays. Moreover, the anti-inflammatory potential of these extracts was demonstrated in an in vitro model of acute intestinal inflammation using differentiated Caco-2 cells. The results showed that all the extracts appeared endowed with excellent antioxidant/free radical scavenging properties. In particular, the extracts from both R. algeriensis and R. tunetanus flowers, and that from R. algeriensis stems were characterized by a remarkable SOD-like and NO-scavenging activity, as well as by the capability to protect albumin against HClO-induced degradation. Furthermore, the extracts from flowers of both Rumex species, as well as R. algeriensis stems, showed an anti-inflammatory effect in intestinal epithelial cells, as demonstrated by the inhibition of TNF-α-induced gene expression of IL-6 and IL-8. In conclusion, R. algeriensis and R. tunetanus have shown to be potential sources of bioactive products to be used in the prevention and treatment of pathologies related to oxidative stress and inflammation.

Use of ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry system as valuable tool for an untargeted metabolomic profiling of Rumex tunetanus flowers and stems and contribution to the antioxidant activity.

Rumex tunetanus is an untapped resource with no available information about their chemical composition. That is why, the present study has conducted comprehensive metabolites profiling of the hydromethanol extracts of the Rumex tunetanus flowers and stems using RP-UHPLC-ESI-QTOF-MS. As results, 60 compounds were characterized for the first time in Rumex tunetanus between them, 18 photochemical were firstly identified in the Polygonaceae family in negative ionization mode. In this regard, quercetin-3-O-glucuronide and (-)-epicatechin gallate were the most abundant phenolic compounds in flowers and stems, respectively. Moreover, positive correlations were found between the antioxidant activity measured by DPPH and FRAP assays with the total phenolic compounds (r = 0.98; r = 0.99, respectively) and the abundance of some phenolic subfamilies such as hydroxycinnamic acids, hydroxybenzoic acids, flavonols and flavones with r > 0.86. The compounds displaying significant (P < 0.01) and good correlations with the antioxidant activity (r > 0.93) were hydroxybenzoic acid, rutin, quercetin-3-O-glucuronide, quercetin-3-O-glucoside, quercetin and luteolin-7-O-rutinoside. In conclusion, Rumex tunetanus flowers and stems showed different bioactive compound profiles and significant antioxidant properties and could be further used in food and nutraceutical industries.

Antibacterial screening of Rumex species native to the Carpathian Basin and bioactivity-guided isolation of compounds from Rumex aquaticus.

Plants belonging to the genus Rumex (family Polygonaceae) are used worldwide in traditional medicine for the treatment of various diseases caused by different microorganisms (e.g. bacteria-related dermatologic conditions, dysentery and enteritis). The present study focused on the antibacterial screening of Rumex species native to the Carpathian Basin, and isolation of compounds from one of the most efficient species, Rumex aquaticus. The antibacterial effects of n-hexane, chloroform and aqueous fractions of methanol extracts prepared from different parts of 14 Rumex species (R. acetosella, R. acetosa, R. alpinus, R. aquaticus, R. conglomeratus, R. crispus, R. hydrolapathum, R. obtusifolius subsp. obtusifolius, R. obtusifolius subsp. subalpinus, R. patientia, R. pulcher, R. scutatus, R. stenophyllus and R. thyrsiflorus) were investigated against Staphylococcus epidermidis, S. aureus, MRSA, Bacillus subtilis, Moraxella catarrhalis, Streptococcus pyogenes, S. pneumoniae, S. agalactiae, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae using the disc diffusion method. Mainly the n-hexane and chloroform extracts prepared from the roots of the plants displayed high antibacterial activity (inhibition zones>15mm) against one or more bacterial strains. The highly active extracts of the aerial part and root of R. aquaticus were subjected to a multistep separation procedure. 19 Compounds, among them naphthalenes (musizin, and its glucoside, torachrysone-glucoside, 2-methoxystypandrone), anthraquinones (emodin, chrysophanol, physcion, citreorosein, chrysophanol-8-O-glucoside), flavonoids (quercetin, quercetin-3,3'-dimethylether, isokaempferide, quercetin 3-O-arabinoside, quercetin 3-O-galactoside, catechin), stilbenes (resveratrol, piceid), and 1-stearoylglycerol were isolated from the plant. The antibacterial activities of isolated compounds were determined, and it was observed that especially naphthalenes exerted remarkable antibacterial effects against several bacterial strains.

Hybridization between alien species Rumex obtusifolius and closely related native vulnerable species R. longifolius in a mountain tourist destination.

Alien species expand their distribution by transportation network development. Hybridization between alien species Rumex obtusifolius and closely related native vulnerable species R. longifolius was examined in a mountain tourist destination in central Japan. The three taxa were morphologically identified in the field. Stem height and leaf area were greater in R. longifolius than R. obtusifolius; hybrids were intermediate between the two Rumex species. R. longifolius and the hybrids grew mainly in wet land and the river tributary; R. obtusifolius grew mainly at the roadside and in meadows. Hybrid germination rates of pollen and seeds were much lower than for the two Rumex species. Clustering analysis showed the three taxa each formed a cluster. Most hybrids were F1 generation; the possibility was low of introgression into the two Rumex species by backcross. This study clarified that (1) hybridization occurred between R. obtusifolius and R. longifolius because they occurred together in a small area, but grew in different water habitat conditions, and (2) hybridization was mostly F1 generation because hybrid pollen and seed fertility was low. However, we need caution about introgression into R. longifolius by R. obtusifolius in this area because of the slight possibility of F2 generation and backcrosses.

Contrasting patterns of transposable element and satellite distribution on sex chromosomes (XY1Y2) in the dioecious plant Rumex acetosa.

Rumex acetosa is a dioecious plant with the XY1Y2 sex chromosome system. Both Y chromosomes are heterochromatic and are thought to be degenerated. We performed low-pass 454 sequencing and similarity-based clustering of male and female genomic 454 reads to identify and characterize major groups of R. acetosa repetitive DNA. We found that Copia and Gypsy retrotransposons dominated, followed by DNA transposons and nonlong terminal repeat retrotransposons. CRM and Tat/Ogre retrotransposons dominated the Gypsy superfamily, whereas Maximus/Sireviruses were most abundant among Copia retrotransposons. Only one Gypsy subfamily had accumulated on Y1 and Y2 chromosomes, whereas many retrotransposons were ubiquitous on autosomes and the X chromosome, but absent on Y1 and Y2 chromosomes, and others were depleted from the X chromosome. One group of CRM Gypsy was specifically localized to centromeres. We also found that majority of previously described satellites (RAYSI, RAYSII, RAYSIII, and RAE180) are accumulated on the Y chromosomes where we identified Y chromosome-specific variant of RAE180. We discovered two novel satellites-RA160 satellite dominating on the X chromosome and RA690 localized mostly on the Y1 chromosome. The expression pattern obtained from Illumina RNA sequencing showed that the expression of transposable elements is similar in leaves of both sexes and that satellites are also expressed. Contrasting patterns of transposable elements (TEs) and satellite localization on sex chromosomes in R. acetosa, where not only accumulation but also depletion of repetitive DNA was observed, suggest that a plethora of evolutionary processes can shape sex chromosomes.

Contrasting patterns of genetic divergence in two sympatric pseudo-metallophytes: Rumex acetosa L. and Commelina communis L.

BACKGROUND: Patterns of genetic divergence between populations of facultative metallophytes have been investigated extensively. However, most previous investigations have focused on a single plant species making it unclear if genetic divergence shows common patterns or, conversely, is species-specific. The herbs Rumex acetosa L. and Commelina communis L. are two pseudo-metallophytes thriving in both normal and cupriferous soils along the middle and lower reaches of the Yangtze River in China. Their non-metallicolous and metallicolous populations are often sympatric thus providing an ideal opportunity for comparative estimation of genetic structures and divergence under the selective pressure derived from copper toxicity. RESULTS: In the present study, patterns of genetic divergence of R. acetosa and C. communis , including metal tolerance, genetic structure and genetic relationships between populations, were investigated and compared using hydroponic experiments, AFLP, ISSR and chloroplast genetic markers. Our results show a significant reduction in genetic diversity in metallicolous populations of C. communis but not in R. acetosa . Moreover, genetic differentiation is less in R. acetosa than in C. communis , the latter species also shows a clustering of its metallicolous populations. CONCLUSIONS: We propose that the genetic divergences apparent in R. acetosa and C. communis , and the contrasting responses of the two species to copper contamination, might be attributed to the differences in their intrinsic physiological and ecological properties. No simple and generalised conclusions on genetic divergence in pseudo-metallophytes can thus be drawn.

Molecular phylogeny and systematics of the highly polymorphic Rumex bucephalophorus complex (Polygonaceae).

Rumex bucephalophorus is a very polymorphic species that has been subjected to various taxonomic studies in which diverse infraspecific taxa have been recognised on the basis of diaspore traits. In this study we used molecular markers (ITS and AFLP) to explore this remarkable diversity, to test previous hypotheses of classification, and attempt to explain biogeographic patterns. Results show that R. bucephalophorus forms a monophyletic group in which diversification began around 4.2 Mya, at the end of Messinian Salinity Crisis. The two molecular markers clearly show a deep divergence separating subsp. bucephalophorus from all other subspecific taxa, among which subsp. canariensis also constitutes a separate and well distinguishable unit. In contrast, subspecies hispanicus and subsp. gallicus constitute a monophyletic group in which three subgroups can be recognised: subsp. hispanicus, subsp. gallicus var. gallicus and subsp. gallicus var. subaegeus. However, these three subgroups are not clearly distinguished genetically or morphologically, so that in formal classification it would be preferable to treat them at the varietal level.

A satellite DNA evolutionary analysis in the North American endemic dioecious plant Rumex hastatulus (Polygonaceae).

We studied the evolution of RAE180 satellite DNA family in the North American endemic dioecious plant Rumex hastatulus. In this species, the Texas race is characterized by a single XX/XY sex chromosome system, whereas the North Carolina race has evolved a derived complex XX/XY(1)Y(2) sex chromosome system. RAE180 repeats were autosomic and poorly represented (2 × 10(-4)% of the genome) with no differences between individuals of different genders or different races of R. hastatulus. In fact, the sex chromosomes of the North Carolina race are still euchromatic, and they have not accumulated satellite DNA sequences, which contrasts with that occurring in the rest of dioecious XX/XY(1)Y(2) Rumex species. In R. hastatulus, we detected the existence of three RAE180 subfamilies. Notwithstanding, while in the Texas race the TX1/NC1 subfamily is the most frequent, the TX2/NC2 subfamily is the most abundant in the North Carolina race. Additionally, the third, less represented subfamily (TX3/NC3) appears currently as relict sequences in both genomes. A common feature of RAE180 satellite is the sudden replacement of one sequence variant by another in different species (or populations as in R. hastatulus races). Thus, the phylogenetic analysis of RAE180 repeats from six dioecious Rumex species supports the "library" hypothesis. According to this hypothesis, we assume that a set of divergent RAE180 variants were present in the ancestral genome of dioecious Rumex species, from which novel tandem arrays originated by the amplification of different variants in different lineages. Differential levels of RAE180 satellite DNA amplification in each lineage, at different evolutionary times, and in different chromosomal positions gave rise to differential patterns of sequence evolution.

Characterization of RUSI, a telomere-associated satellite DNA, in the genus Rumex (Polygonaceae).

A satellite-DNA family (RUSI) has been isolated and characterized in Rumexinduratus Boiss and Reuter (Polygonaceae), an Iberian endemic polygamous sorrel. The RUSI repeats are 170 bp in length and approximately 68% AT-rich containing different variants of degenerate telomere motifs--(TT)(n)AN(GG)(n) -, a typical feature of subtelomeric DNA repeats adjacent to telomeres, which have been referred to as telomere-associated sequences or TASs. In fact, fluorescent in situhybridization showed that this satellite DNA is located in subtelomeric positions of most of the chromosomes of R. induratus, with some centromeric loci. PCR and Southern-blot hybridization assays for sequence conservation in the genus Rumex, indicated that the RUSI sequences are restricted to the genomes of R. induratus and R. scutatus, both species of the section Scutati, suggesting that they are recently evolved. Sequence variation within the two species is high (mean value of sequence differences between repeats of 15% for R. induratus and 7.5% for R. scutatus) and the degree of sequence differentiation between species is low with no species-specific variants, postulated to be due to slowed rates of spreading of sequence variants by molecular homogenizing mechanisms. Characteristics of RUSI sequences are discussed in the light of their chromosomal location and analyzed for their evolutionary and phylogenetic implications.

Phytoextraction potential of two Rumex acetosa L. accessions collected from metalliferous and non-metalliferous sites: effect of fertilization.

Metal tolerance and phytoextraction potential of two common sorrel (Rumex acetosa L.) accessions, collected from a Pb/Zn contaminated site (CS, Lanestosa) and an uncontaminated site (UCS, Larrauri), were studied in fertilized and non-fertilized pots prepared by combining soil samples from both sites in different proportions (i.e., 0%, 33%, 66% and 100% of Lanestosa contaminated soil). The original metalliferous mine soil contained 20480, 4950 and 14 mg kg(-1) of Zn, Pb and Cd, respectively. The microcosm experiment was carried out for two months under greenhouse controlled conditions. It was found that fertilization increased mean plant biomass of both accessions as well as their tolerance. However, only the CS accession survived all treatments even though its biomass decreased proportionally according to the percentage of contaminated mine soil present in the pots. This metallicolous accession would be useful for the revegetation and phytostabilization of mine soils. Due to its high concentration and bioavailability in the contaminated soil, the highest values of metal phytoextracted corresponded to Zn. The CS accession was capable of efficiently phytoextracting metal from the 100% mine soil, indeed reaching very promising phytoextraction rates in the fertilized pots (6.8 mg plant(-1) month(-1)), similar to the ones obtained with hyperaccumulator plants. It was concluded that fertilization is certainly worth being considered for phytoextraction and revegetation with native plants from metalliferous soils.

The evolution of sex chromosomes in the genus Rumex (Polygonaceae): Identification of a new species with heteromorphic sex chromosomes.

The structural features and evolutionary state of the sex chromosomes of the XX/XY species of Rumex are unknown. Here, we report a study of the meiotic behaviour of the XY bivalent in Rumex acetosella and R. suffruticosus, a new species which we describe cytogenetically for the first time in this paper, and also that of the XY(1)Y(2) trivalent of R. acetosa by both conventional cytogenetic techniques and analysis of synaptonemal complex formation. Fluorescent in situ hybridization with satellite DNA and rDNA sequences as probes was used to analyse the degree of cytogenetic differentiation between the X and Y chromosomes in order to depict their evolutionary stage in the three species. Contrasting with the advanced state of genetic differentiation between the X and the Y chromosomes in R. acetosa, we have found that R. acetosella and R. suffruticosus represent an early stage of genetic differentiation between sex chromosomes. Our findings further demonstrate the usefulness of the genus Rumex as a model for analysing the evolution of sex chromosomes in plants, since within this genus it is now possible to study the different levels of genetic differentiation between the sex chromosomes and to analyse their evolutionary history from their origin.

Excessive variation in Y chromosomal DNA in Rumex acetosa (Polygonaceae).

Rumex acetosa is one of the few angiosperms that possesses sex chromosomes. The same types of abundant repetitive sequences cover both heterochromatic Y chromosomes present in males. The aim of this study was to investigate genetic variation in paternally inherited Y chromosomal DNA and in maternally inherited cpDNA, and to find out whether the examined genomic regions are suited to a phylogeographic study in R. acetosa. DNA sequence polymorphisms present in the 850-bp heterochromatic segment on the Y chromosomes were compared to variation in the 409-bp long chloroplast section (trnL- trnF spacer) in R. acetosa originating from several European locations and from the Altai mountains in Russia. A great amount of genetic variation was detected within the Y chromosomal region while only four chloroplast genotypes were detected. Although the chloroplast haplotypes possessed some geographic pattern, no clear phylogeographic pattern was detected based on the variable Y chromosomes. The mean Y chromosomal nucleotide diversity among all samples equaled 6.6 %, and the mean proportion of polymorphic sites per individual equaled 8.2 % among SNP sites and 1.7 % among all sites investigated. The high number of substitutions detected in the Y chromosomal DNA shows that this heterochromatic sequence has a high mutation rate. The diversity pattern indicates that gene flow via pollen is extensive and it blurs any geographical pattern in the Y chromosomal variation. The high number of repeats and uncertainty concerning the extent of recombination between the two Y chromosomes impair the usability of the Y chromosomal segment for phylogeographic or population genetic studies.

The origin and evolution of the variability in a Y-specific satellite-DNA of Rumex acetosa and its relatives.

In this paper, we analyze a satellite-DNA family, the RAYSI family, which is specific of the Y chromosomes of Rumex acetosa, a dioecious plant species with a multiple sex-chromosome system in which the females are XX and the males are XY(1)Y(2). Here, we demonstrate that this satellite DNA is common to other relatives of R. acetosa, including Rumex papillaris, Rumex intermedius, Rumex thyrsoides and Rumex tuberosus that are also dioecious species with a multiple system of sex chromosomes. This satellite-DNA family is absent from the genomes of other dioecious Rumex species having an XX/XY sex-chromosome system. Our data confirm recent molecular phylogenies that support a unique origin for all dioecious species of Rumex and two separate lineages for species with single or complex sex-chromosome systems. Our data also support an accelerated degeneration of Y-chromosome in XX/XY(1)Y(2) species by the accumulation of satellite-DNA sequences. On the other hand, the particular non-recombining nature of the Y chromosomes of R. acetosa and their closest relatives lead to a particular mode of evolution of RAYSI sequences. Thus, mechanisms leading to the suppression of recombination between the Y chromosomes reduced the rate of concerted evolution and gave rise to the apparition of different RAYSI subfamilies. Thus, R. acetosa and R. intermedius have two subfamilies (the RAYSI-S and RAYSI-J subfamilies and the INT-A and INT-B subfamilies, respectively), while R. papillaris only has one, the RAYSI-J subfamily. The RAYSI-S and RAYSI-J subfamilies of R. acetosa differ in 83 fixed diagnostic sites and several diagnostic deletions while the INT-A and the INT-B of R. intermedius differ in 27 fixed diagnostic sites. Pairwise comparisons between RAYSI-S and RAYSI-J sequences or between INT-A and INT-B sequences revealed these sites to be shared mutations detectable in repeats of the same variant in same positions. Evolutionary comparisons suggest that the subfamily RAYSI-J has appeared in the common ancestor of R. acetosa and R. papillaris, in which RAYSI-J has replaced totally (R. papillaris) or almost totally the ancestral sequence (R. acetosa). This scenario assumes that RAYSI-S sequences should be considered ancestral sequences and that a secondary event of subfamily subdivision should be occurring in R. intermedius, with their RAYSI subfamilies more closely related to one another than with other RAYSI sequences. Our analysis suggests that the different subfamilies diverged by a gradual and cohesive way probably mediated by sister-chromatid interchanges while their expansion or contraction in number might be explained by alternating cycles of sudden mechanisms of amplification or elimination.

The evolution of reproductive systems and sex-determining mechanisms within rumex (polygonaceae) inferred from nuclear and chloroplastidial sequence data.

The genus Rumex includes hermaphroditic, polygamous, gynodioecious, monoecious, and dioecious species, with the dioecious species being represented by different sex-determining mechanisms and sex-chromosome systems. Therefore, this genus represents an exceptional case study to test several hypotheses concerning the evolution of both mating systems and the genetic control of sex determination in plants. Here, we compare nuclear intergenic transcribed spacers and chloroplast intergenic sequences of 31 species of Rumex. Our phylogenetic analysis supports a systematic classification of the genus, which differs from that currently accepted. In contrast to the current view, this new phylogeny suggests a common origin for all Eurasian and American dioecious species of Rumex, with gynodioecy as an intermediate state on the way to dioecy. Our results support the contention that sex determination based on the balance between the number of X chromosomes and the number of autosomes (X/A balance) has evolved secondarily from male-determining Y mechanisms and that multiple sex-chromosome systems, XX/XY1Y2, were derived twice from an XX/XY system. The resulting phylogeny is consistent with a classification of Rumex species according to their basic chromosome number, implying that the evolution of Rumex species might have followed a process of chromosomal reduction from x = 10 toward x = 7 through intermediate stages (x = 9 and x = 8).