Anthyllis Vulneraria Research Articles

DEMOGRAPHY OF ALPINE SHORT-LIVED PLANTS, LONGEVITY AND ONTOGENY STAGE DURATIONS

The aim - to evaluate lifespan (full cycle) and ontogeny stage durations of nine alpine short-lived North- West Caucasus plants. Methods. For calculation we used a new method which was developed and suggested earlier by us. This method is based on a discrete ontogeny description and on the probability theory and random processes. The data on the monitoring of the marked individuals were collected during six years. Results. We found out that the lifespan of Anthyllis vulneraria is 2.6±0.3 years (hereinafter “±” is Standard error), Draba hispida – 4.5±0.3, Murbeckiella huetii – 4.6±1.1, Carum meifolium – 7.8±1.4, Eritrichium caucasicum – 9.1±1.4, Trifolium badium – 10.3±2.6, Sedum tenellum – 11±2.05, Androsace albana – 12.1±2.5, Minuartia recurva – 22.9±4.5. Also we demonstrated the matrix population models for studied plants, which show the probability of transition of individuals from one ontogeny stage to another in time interval (in our experiment – 1 year). Conclusion . Mortality of seedlings and juvenile plants, except Murbeckiella huetii, is around and more than 50%. Two years is the minimal amount of time that is necessary for full cycle of short-lived alpine plants, as it was shown for Anthyllis vulneraria, Murbeckiella huetii и Trifolium badium. A 3-12 years lifespan was calculated for other studied species. Persistence of Eritrichium caucasicum and Androsace albana populations provided by resistance of adult vegetative plants.

Past selection explains differentiation in flowering phenology of nearby populations of a common alpine plant

The timing of and relative investment in reproductive events are crucial fitness determinants for alpine plants, which have limited opportunities for reproduction in the cold and short growing seasons at high elevations. We use the alpine Anthyllis vulneraria to study whether flowering phenology and reproductive allocation have been under diversifying selection, and to assess genetic diversity and plastic responses to drought in these traits. Open-pollinated maternal families from three populations in each of two regions from the Swiss Alps with contrasting precipitation were grown in low and high soil moisture in a common garden. We measured onset, peak, and end of flowering, as well as vegetative and reproductive aboveground biomass. Population differentiation for each character (Q ST) was compared to differentiation at neutral microsatellite loci (F ST) to test for past selection. We found population differentiation in onset and peak of flowering which results from natural selection according to Q ST–F ST. End of flowering and biomass were not significantly differentiated among populations. Reduced soil moisture had no consistent effect on mean onset of flowering, and advanced peak and end of flowering by less than 1 week. Reproductive biomass was strongly decreased by lowered soil moisture. No genetic variation within or among populations was found for plasticity in any trait measured. The results suggest past heterogeneous selection on onset and peak of flowering in alpine Anthyllis vulneraria and potentially indicate local adaptation to differences in snowmelt date over distances <5 km. Limited variation in plastic responses to reduced soil moisture suggests that soil moisture might not vary between populations.

Hidden founder effects: small-scale spatial genetic structure in recently established populations of the grassland specialist plant Anthyllisvulneraria.

The long-term establishment success of founder plant populations has been commonly assessed based on the measures of population genetic diversity and among population genetic differentiation, with founder populations expected to carry sufficient genetic diversity when population establishment is the result of many colonists from multiple source populations (the 'migrant pool' colonization model). Theory, however, predicts that, after initial colonization, rapid population expansion may result in a fast increase in the extent of spatial genetic structure (SGS), independent of extant genetic diversity. This SGS can reduce long-term population viability by increasing inbreeding. Using 12 microsatellite markers, we inferred colonization patterns in four recent populations of the grassland specialist plant Anthyllisvulneraria and compared the extent of SGS between recently established and old populations. Assignment analyses of the individuals of recent population based on the genetic composition of nine adjacent putative source populations suggested the occurrence of the 'migrant pool' colonization model, further confirmed by high genetic diversity within and low genetic differentiation among recent populations. Population establishment, however, resulted in the build-up of strong SGS, most likely as a result of spatially restricted recruitment of the progeny of initial colonists. Although reduced, significant SGS was nonetheless observed to persist in old populations. The presence of SGS was in all populations associated with elevated inbreeding coefficients, potentially affecting the long-term viability of these populations. In conclusion, this study illustrates the importance of studying SGS next to population genetic diversity and differentiation to adequately infer colonization patterns and long-term establishment success of plant species.

Rhizobium metallidurans sp. nov., a symbiotic heavy metal resistant bacterium isolated from the Anthyllis vulneraria Zn-hyperaccumulator.

A Gram-stain-negative, aerobic, rod-shaped, non-spore-forming bacterium (ChimEc512(T)) was isolated from 56 host seedlings of the hyperaccumulating Anthyllis vulneraria legume, which was on an old zinc mining site at Les Avinières, Saint-Laurent-Le-Minier, Gard, South of France. On the basis of 16S rRNA gene sequence similarities, strain ChimEc512(T) was shown to belong to the genus Rhizobium and to be most closely related to Rhizobium endophyticum CCGE 2052(T) (98.4%), Rhizobium tibeticum CCBAU 85039(T) (98.1%), Rhizobium grahamii CCGE 502(T) (98.0%) and Rhizobium mesoamericanum CCGE 501(T) (98.0%). The phylogenetic relationships of ChimEc512(T) were confirmed by sequencing and analyses of recA and atpD genes. DNA-DNA relatedness values of strain ChimEc512(T) with R. endophyticum CCGE 2052(T), R. tibeticum CCBAU 85039(T), R. mesoamericanum CCGE 52(T), Rhizobium grahamii CCGE 502(T), Rhizobium etli CCBAU 85039(T) and Rhizobium radiobacter KL09-16-8-2(T) were 27, 22, 16, 18, 19 and 11%, respectively. The DNA G+C content of strain ChimEc512(T) was 58.9 mol%. The major cellular fatty acid was C18 : 1ω7c, characteristic of the genus Rhizobium . The polar lipid profile included phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and moderate amounts of aminolipids, phospholipid and sulfoquinovosyl diacylglycerol. Although ChimEc512(T) was able to nodulate A. vulneraria, the nodC and nifH genes were not detected by PCR. The rhizobial strain was tolerant to high concentrations of heavy metals: up to 35 mM Zn and up to 0.5 mM Cd and its growth kinetics was not impacted by Zn. The results of DNA-DNA hybridizations and physiological tests allowed genotypic and phenotypic differentiation of strain ChimEc512(T) from species of the genus Rhizobium with validly published names. Strain ChimEc512(T), therefore, represents a novel species, for which the name Rhizobium metallidurans sp. nov. is proposed, with the type strain ChimEc512(T) ( =DSM 26575 = CIP 110550(T)).

Analysis of the variability of nine natural Anthyllis vulneraria s.l. populations

It was established on the basis of examination of nine natural &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; populations from various geographical regions of Poland that the type of variability differs in the coastal, Wielkopolska lowland and Tatra populations. The results of the experiments support the idea of the taxonomic distinctness of the coastal, lowland and montane plants; the coastal populations belong to &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; ssp. maritima, Tatra populations to &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; ssp. &lt;i&gt;affinis&lt;/i&gt; and populations from the Wielkopolska Lowland and Sudetes to &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; ssp. &lt;i&gt;polyphylla&lt;/i&gt; (Kostrakiewicz, 1959).

Variability of enzymatic systems in natural populations of Anthyllis vulneraria s. 1. from three geographic regions of Poland. Part II. Geographic variability of enzymatic systems in six woundwort populations

Differences between populations of &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; s.l. from three distinct geographic regions of Poland for four enzyme systems showed different variability and various patterns of isoenzymes. The smallest differences between populations from similar habitats were found for two populations from dune. Data from statistical analysis of enzyme variation in the examined natural populations of &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; were correlated with data of statistical analyses of morphological features, features of flowers, and analyses of phenolic compounds.

Analysis of the variability of nine natural Anthyllis vulneraria s. l. populations. Part II. Biometry of flowers

Variation of 1l vegetative traits has been studied in 9 natural populations of &lt;i&gt;Anthyllis vulneraria&lt;/i&gt;. The analysis was performed in the field and then, for the second time, on samples of these populations grown in experimental garden in similar conditions. Three genetically different groups has been discovered: l - populations from the Tatra Mountains, 2 - from the Central Poland and Sudetes, 3 - from the Baltic Sea Coast.

Variability of enzymatic systems in natural populations of Anthyllis vulneraria s. 1. from three geographic regions of Poland. Part I. Ontogenetic variability of enzymatic systems in three woundwort populations during plant development

Analysis of variation in the four enzymatic systems of three populations of &lt;i&gt;Anthyllis vulneraria&lt;/i&gt; was made. Different polymorphism of enzyme .protein® in six terms of plant development was found by isoenzyme electrophoresis on polyacrylamide gel. Each population had a specific isoenzyme pattern and specific variability in the terms.

Chromatographic analysis of phenol compounds in six natural populations of Anthyllis vulneraria (L.)

Thin-layer chromatography was used to study the phenol composition in individual plants from six natural populations of &lt;i&gt;Anthyllis&lt;i&gt; collected from three distinct geographic regions of Poland. The results showed a variability of the phenols in the examined populations. The populations from Wielkopolska region proved to be most variable, showing the greatest number of phenols. The lowest number of the phenols studies was found in the Tatry populations. Each population showed its own particular spectrum of phenolic compounds. It was found that the populations originating from similar habitats showed more common spots than those from different regions. Populations from the Tatra region were found to differ most from the rest.

Prospecting metal-tolerant rhizobia for phytoremediation of mining soils from Morocco using Anthyllis vulneraria L.

The aim of this work was using the legume plant Anthyllis vulneraria L. (ecotype metallicolous) as a trap plant, in order to isolate metal-tolerant rhizobial strains from metal-contaminated soils from Morocco, with pollution indexes spanning three orders of magnitude. As bioindicator, soil bacterial density was inversely correlated to the pollution index. Forty-three bulk soil bacteria and sixty two bacteria from nodules were isolated. The resistance of bacteria from nodules to heavy metals was four to ten times higher than that of bulk soil bacteria, reaching high maximum tolerable concentrations for Cd (2mM), Cu (2mM), Pb (7mM), and Zn (3mM). Besides, some strains show multiple metal-tolerant abilities and great metal biosorption onto the bacterial surface. Amplification and restriction analysis of ribosomal 16S rDNA (ARDRA) and 16S ribosomal DNA (rDNA) sequencing were used to assess biodiversity and phylogenetic position among bacteria present in nodules. Our results suggest that a great diversity of non-rhizobial bacteria (alpha- and gamma-proteobacteria) colonize nodules of Anthyllis plants in contaminated soils. Taking together, our results evidence that, in polluted soils, rhizobia can be displaced by non-rhizobial (and hence, non-fixing) strains from nodules. Thus, the selection of metal-resistant rhizobia is a key step for using A. vulneraria symbioses for in situ phytoremediation.

The leguminous species Anthyllis vulneraria as a Zn-hyperaccumulator and eco-Zn catalyst resources.

Anthyllis vulneraria was highlighted here as a Zn-hyperaccumulator for the development of a pilot phytoextraction process in the mine site of Les Avinières in the district of Saint-Laurent-Le-Minier. A. vulneraria appeared to hyperaccumulate the highest concentration of Zn in shoots with a better metal selectivity relative to Cd and Pb than the reference Zn-hyperaccumulator Noccea caerulescens. A bigger biomass production associated to a higher Zn concentration conducted A. vulneraria to the highest total zinc gain per hectare per year. As a legume, A. vulneraria was infected by rhizobia symbionts. Inoculation of A. vulneraria seeds showed a positive impact on Zn hyperaccumulation. A large-scale culture process of symbiotic rhizobia of A. vulneraria was investigated and optimized to allow large-scale inoculation process. Contaminated shoots of A. vulneraria were not considered as wastes and were recovered as Eco-Zn catalyst in particular, examples of organic synthesis, electrophilic aromatic substitution. Eco-Zn catalyst was much more efficient than conventional catalysts and allowed greener chemical processes.

Growth, photosynthetic acclimation and yield quality in legumes under climate change simulations: An updated survey

Continued emissions of CO2, derived from human activities, increase atmospheric CO2 concentration. The CO2 rise stimulates plant growth and affects yield quality. Effects of elevated CO2 on legume quality depend on interactions with N2-fixing bacteria and mycorrhizal fungi. Growth at elevated CO2 increases photosynthesis under short-term exposures in C3 species. Under long-term exposures, however, plants generally acclimate to elevated CO2 decreasing their photosynthetic capacity. An updated survey of the literature indicates that a key factor, perhaps the most important, that characteristically influences this phenomenon, its occurrence and extent, is the plant source–sink balance. In legumes, the ability of exchanging C for N at nodule level with the N2-fixing symbionts creates an extra C sink that avoids the occurrence of photosynthetic acclimation. Arbuscular mycorrhizal fungi colonizing roots may also result in increased C sink, preventing photosynthetic acclimation. Defoliation (Anthyllis vulneraria, simulated grazing) or shoot cutting (alfalfa, usual management as forage) largely increases root/shoot ratio. During re-growth at elevated CO2, new shoots growth and nodule respiration function as strong C sinks that counteracts photosynthetic acclimation. In the presence of some limiting factor, the legumes response to elevated CO2 is weakened showing photosynthetic acclimation. This survey has identified limiting factors that include an insufficient N supply from bacterial strains, nutrient-poor soils, low P supply, excess temperature affecting photosynthesis and/or nodule activity, a genetically determined low nodulation capacity, an inability of species or varieties to increase growth (and therefore C sink) at elevated CO2 and a plant phenological state or season when plant growth is stopped.

Metal accumulation strategies in plants spontaneously inhabiting Zn-Pb waste deposits

Metal (Zn, Pb, Cd, Cu, Ni, Cr) accumulation in shoots of 38 plant species spontaneously colonizing three Zn-Pb waste deposits in southern Poland was studied in order to find out if the age of the waste (30–130 years) or its type (slag or flotation residues) influence metal content in plants and to identify species potentially suitable for biomonitoring and phytoremediation. The total metal concentrations in the waste upper layers ranged from 7300 to 171790mgkg−1 for Zn, from 1390 to 22265mgkg−1 for Pb, and from 66 to 1464mgkg−1 for Cd, whereas CaCl2-extracted fractions accounted for 0.034–0.11 %, 0.005–0.03 %, and 0.28–0.62 % of total Zn, Pb and Cd concentrations, respectively. The concentrations of Cu, Ni, and Cr in substrates and in plants were low and ranged within the background values. Metal accumulation in plant shoots was poorly correlated with both total and CaCl2-extracted forms of metals in the substrate and was highly variable among species and also specimens of the same species. The highest mean concentrations of Zn, Pb and Cd were found in Anthyllis vulneraria L. (901.5mgkg−1), Echium vulgare L. (116.92mgkg−1), and Hieracium piloselloides Vill. (26.86mgkg−1), respectively. Besides Reseda lutea L., no species appeared to be a good indicator of polymetallic environment pollution based on chemical analysis of shoots; however, metal accumulation in the whole plant communities of a particular contaminated area might be an accurate tool for assessment of metal transfer to vegetation irrespective of the type or age of the waste. All the species studied developed a metal exclusion strategy, thus exhibiting potential for phytostabilization of metalliferous wastelands.

CadA of Mesorhizobium metallidurans isolated from a zinc-rich mining soil is a PIB-2-type ATPase involved in cadmium and zinc resistance

Mesorhizobium metallidurans STM 2683T is a nitrogen-fixing bacterium that nodulates Anthyllis vulneraria in mine tailings highly contaminated in zinc, lead and cadmium. To study the mechanisms whereby this bacterium copes with metals, we functionally screened a cosmid genomic library of M. metallidurans for zinc or cadmium tolerance. A cosmid clone harbored a gene encoding PIB-type ATPase homologous to CadA that leads to cadmium and zinc resistance in Escherichia coli. The CadA protein structure presents one duplication of the two N-terminal metal binding domains (i.e. a heavy metal-associated domain followed by a histidine-rich domain) which allows specific binding to zinc and cadmium cations. A cadA-deleted strain of M. metallidurans failed to grow at high zinc concentrations (2mM) and its growth was delayed at lower zinc concentrations. Expression studies using a transcriptional fusion of cadA promoter to gfp showed that cadA is specifically induced in a dose-dependent manner by zinc and cadmium in M. metallidurans in vitro conditions and into A. vulneraria nodules after Zn stress. Metal induction sensitivity was increased in the strain where cadA gene was deleted.This study identified cadA as a first mesorhizobial resistance determinant involved in detoxification of cadmium and zinc and which confers upon M. metallidurans greater capacity for coping with high zinc concentrations. This improves the knowledge of this bacterium for potential use as a symbiotic inoculant of Anthyllis in phytostabilization strategies of metal-rich sites.

New microsatellite markers for Anthyllis vulneraria (Fabaceae), analyzed with Spreadex gel electrophoresis.

• Premise of the study: New microsatellite primers were developed for the diploid herb Anthyllis vulneraria. These primers will be used in upcoming studies focusing on random genetic variation, local adaptation, and phenotypic plasticity in alpine plants.• Methods and Results: The new primers were adjusted to separate PCR amplicons (70 to 170 bp) on precast Spreadex gels using horizontal gel electrophoresis. No capillary sequencer was needed. Three to twelve alleles were found per locus depending on the population studied.• Conclusions: Our preliminary results showed that the three studied alpine populations are predominantly outcrossing, but include variable levels of self-fertilization.

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn, Pb and Cd mine tailings

Background and aims The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Genome-wide transcriptional responses of two metal-tolerant symbiotic Mesorhizobium isolates to Zinc and Cadmium exposure

BackgroundMesorhizobium metallidurans STM 2683T and Mesorhizobium sp. strain STM 4661 were isolated from nodules of the metallicolous legume Anthyllis vulneraria from distant mining spoils. They tolerate unusually high Zinc and Cadmium concentrations as compared to other mesorhizobia. This work aims to study the gene expression profiles associated with Zinc or Cadmium exposure and to identify genes involved in metal tolerance in these two metallicolous Mesorhizobium strains of interest for mine phytostabilization purposes.ResultsThe draft genomes of the two Mezorhizobium strains were sequenced and used to map RNAseq data obtained after Zinc or Cadmium stresses. Comparative genomics and transcriptomics allowed the rapid discovery of metal-specific or/and strain-specific genes. Respectively 1.05% (72/6,844) and 0.97% (68/6,994) predicted Coding DNA Sequences (CDS) for STM 2683 and STM 4661 were significantly differentially expressed upon metal exposure. Among these, a significant number of CDS involved in transport (13/72 and 13/68 for STM 2683 and STM 4661, respectively) and sequestration (15/72 and 16/68 for STM 2683 and STM 4661, respectively) were identified. Thirteen CDS presented homologs in both strains and were differentially regulated by Zinc and/or Cadmium. For instance, several PIB-type ATPases and genes likely to participate in metal sequestration were identified. Among the conserved CDS that showed differential regulation in the two isolates, we also found znuABC homologs encoding for a high affinity ABC-type Zinc import system probably involved in Zinc homeostasis. Additionally, global analyses suggested that both metals also repressed significantly the translational machinery.ConclusionsThe comparative RNAseq-based approach revealed a relatively low number of genes significantly regulated in the two Mesorhizobium strains. Very few of them were involved in the non-specific metal response, indicating that the approach was well suited for identifying genes that specifically respond to Zinc and Cadmium. Among significantly up-regulated genes, several encode metal efflux and sequestration systems which can be considered as the most widely represented mechanisms of rhizobial metal tolerance. Downstream functional studies will increase successful phytostabilization strategies by selecting appropriate metallicolous rhizobial partners.

Zinc accumulation patterns in four Anthyllis vulneraria subspecies supplemented with mineral nitrogen or grown in the presence of their symbiotic bacteria

Aims This work examines Zn accumulation in four Anthyllis vulneraria subspecies supplemented with mineral nitrogen or grown in the presence of their symbiotic bacteria. Methods Anthyllis vulneraria subspecies were grown hydroponically in the presence of high levels of ZnSO4. The plants were either grown in symbiosis with one of two non-metallicolous or metallicolous Mesorhizobium inoculants or in the presence of KNO3. Results When exposed to 1,000 μM Zn, shoot and root biomass of three out of our four Anthyllis subspecies cultivated with NO3 dropped significantly by about 24–28 %; carpatica, the fourth subspecies, was not affected. Subspecies carpatica Zn tolerance was confirmed when in symbiosis with the metallicolous strain. In the presence of 1,000 μM Zn, the different Anthyllis subspecies concentrated more Zn in their roots than in their shoots and only subsp. carpatica accumulated a significant amount of Zn in its shoots. The most remarkable feature was the drastic decrease in Zn concentration in both roots (up to 2.5–3 fold) and shoots (2.6-fold) of subsp. carpatica exposed to 1,000 μM Zn and nodulated whatever the Mesorhizobium strain used, compared to the N-grown plants.

Rhizostabilzation of a mine tailing higly contaminated: Previous study of Cd localization and speciation inAnthyllis vulneraria

The plant Anthyllis vulneraria has been identified in mine tailings and the aim of this study is to determine the mechanisms developed by the plant and its symbiotic association Mesorhizobium metallidurans to tolerate Cd. We particulary intend to determine the distribution and speciation of Cd in plant using a combination of μXRF (X-ray fluorescence) and Cd XANES and μXANES (X-ray Absorption Near Edge Structure). © Owned by the authors, published by EDP Sciences, 2013.

Local Adaptation of Metallicolous and Non‐Metallicolous Anthyllis vulneraria Populations: Their Utilization in Soil Restoration

Restoration of metalliferous mine soils requires using plant species tolerant to high metal concentrations and adapted to nutrient‐poor soil. Legumes can increase plant productivity through N2‐fixation, but they are often scarce in metalliferous sites. We examined survival, growth, and tolerance of four populations of a legume, Anthyllis vulneraria, from two metalliferous (MET) Zn‐Pb mine sites, Avinières (AV) ([Zn‐EDTA] = 26,000 mg/kg) and Eylie (EY) ([Zn‐EDTA] = 4,632 mg/kg), and two non‐metalliferous (NMET) sites located in the south of France with the aim to select the most appropriate populations for restoration of mined soils. In a common garden experiment, plants from each population were reciprocally grown in soil from the provenance of each population. The two NMET populations exhibited high mortality and low growth rates in soil from the mined sites. The AV MET exhibited a high growth rate in metalliferous soils, but showed high mortality in non‐metalliferous soils. The growth of the EY MET was very low in the AV‐contaminated soil, but was the highest of all populations in moderately and non‐metalliferous soils. Plants from the AV MET population showed a high growth and survival in metalliferous soil and would be appropriate in the restoration of metal‐contaminated sites (&gt;30,000 mg Zn kg−1). The EY MET population would be adapted to the restoration of moderate metal‐contaminated soils (&lt;30,000 mg Zn kg−1). Taking into account the broad distribution of A. vulneraria, these two populations could be suitable for the restoration of derelict mine sites in mediterranean and temperate regions of Europe and North America.