Effect of plant root exudates on the desorption of hexachlorocyclohexane isomers from contaminated soils.

Plants and their associated microbiota can have a significant impact on the behaviour of soil contaminants. Particularly, root exudation is one of the most important plant-associated processes in this respect, as it may have a substantial effect on the bioavailability of soil contaminants, specially of hydrophobic contaminants strongly sorbed by soil. The aim of the present study was to evaluate the effect of root exudates (natural and artificial) on the desorption of α-, ß-, δ- and γ-isomers of hexachlorocyclohexane (HCH) from contaminated soil, using batch experiments. Natural root exudates were obtained from Holcus lanatus plants growing in the same (contaminated) area. Fifteen compounds (mainly organic acids and phenolic compounds) usually found in root exudates were also tested, individually or as mixtures (1 and 10 mM). Both natural and artificial exudates favoured the mobilization of sorbed HCH in soil. The effect was highly significant for α-, ß- and γ-HCH isomers, for which the desorption rates increased by 23.0, 26.8 and 15.5% in the presence of natural root exudates and by 40.1, 25.9 and 25.6% in the presence of the artificial mixture (at 10 mM). The δ-HCH desorption rates increased by less than 10%. The effect of individual exudate components was very variable, but increased with the carbon content, reflecting the significance of hydrophobic interactions between the exudates and HCH molecules in the desorption of these last from soil. These findings indicate that plants may significantly influence the bioavailability of persistent contaminants, with major implications for improving phyto- and bioremediation procedures.

Canopy chlorophyll fluorescence applied to stress detection using an easy-to-build micro-lidar.

LEDFLEX is a micro-lidar dedicated to the measurement of vegetation fluorescence. The light source consists of 4 blue Light-Emitting Diodes (LED) to illuminate part of the canopy in order to average the spatial variability of small crops. The fluorescence emitted in response to a 5-µs width pulse is separated from the ambient light through a synchronized detection. Both the reflectance and the fluorescence of the target are acquired simultaneously in exactly the same field of view, as well as the photosynthetic active radiation and air temperature. The footprint is about 1 m2 at a distance of 8 m. By increasing the number of LEDs longer ranges can be reached. The micro-lidar has been successfully applied under full sunlight conditions to establish the signature of water stress on pea (Pisum Sativum) canopy. Under well-watered conditions the diurnal cycle presents an M shape with a minimum (Fmin) at noon which is Fmin > Fo. After several days withholding watering, Fs decreases and Fmin < Fo. The same patterns were observed on mint (Menta Spicata) and sweet potatoes (Ipomoea batatas) canopies. Active fluorescence measurements with LEDFLEX produced robust fluorescence yield data as a result of the constancy of the excitation intensity and its geometry fixity. Passive methods based on Sun-Induced chlorophyll Fluorescence (SIF) that uses high-resolution spectrometers generate only flux data and are dependent on both the 3D structure of vegetation and variable irradiance conditions along the day. Parallel measurements with LEDFLEX should greatly improve the interpretation of SIF changes.

Speciation and uptake of antimony and arsenic by two populations of Pteris vittata L. and Holcus lanatus L. from co-contaminated soil.

This study aimed to investigate the Sb and As co-accumulating processes of Pteris vittata under soil culture condition, including the transformation of Sb and As, and the difference in co-accumulating ability among different plant species/populations. Two populations of P. vittata and one population of As-tolerant species Holcus lanatus L. were grown on soil co-contaminated by Sb and As. Sb and As speciation in plants was assessed by X-ray absorption near-edge structure (XANES) spectroscopy. P. vittata displayed strong As- but limited Sb-accumulating ability, with the highest shoot concentrations of As and Sb reaching 455 and 26 mg kg-1, respectively. After 28 days culture, the concentrations of Sb and As in the soil solution were reduced by up to 22% and 36% in the P. vittata treatments, respectively. Holcus lanatus showed limited uptake for both metalloids. In P. vittata, the reduction of arsenate to arsenite occurred (with As in shoots all existing as arsenite), but limited reduction of antimonate to antimonite (with more than 90% of Sb in shoots existing as antimonate) was observed. In terms of the differences in metalloid uptake between the two P. vittata populations, the population from the habitat with higher soil As concentration showed 35% higher As uptake than the population from the habitat with lower As concentration. This populational difference may partly result from varying As transformation efficiencies. However, no significant difference was observed in Sb accumulation between the two populations.

Soil-to-plant transfer of arsenic and phosphorus along a contamination gradient in the mining-impacted Ogosta River floodplain.

Riverine floodplains downstream of active or former metal sulfide mines are in many cases contaminated with trace metals and metalloids, including arsenic (As). Since decontamination of such floodplains on a large scale is unfeasible, management of contaminated land must focus on providing land use guidelines or even restrictions. This should be based on knowledge about how contaminants enter the food chain. For As, uptake by plants may be an important pathway, but the As soil-to-plant transfer under field conditions is poorly understood. Here, we investigated the soil-to-shoot transfer of As and phosphorus (P) in wild populations of herbaceous species growing along an As contamination gradient across an extensive pasture in the mining-impacted Ogosta River floodplain. The As concentrations in the shoots of Trifolium repens and Holcus lanatus reflected the soil contamination gradient. However, the soil-to-shoot transfer factors (TF) were fairly low, with values mostly below 0.07 (TF=Asshoot/Assoil). We found no evidence for interference of As with P uptake by plants, despite extremely high molar As:P ratios (up to 2.6) in Olsen soil extracts of the most contaminated topsoils (0-20cm). Considering the restricted soil-to-shoot transfer, we estimated that for grazing livestock As intake via soil ingestion is likely more important than intake via pasture herbage.

Plants increase arsenic in solution but decrease the non-specifically bound fraction in the rhizosphere of an alkaline, naturally rich soil.

We aimed at determining the major physical-chemical processes that drive arsenic (As) dynamic in the rhizosphere of four species (Holcus lanatus, Dittrichia viscosa, Lotus corniculatus, Plantago lanceolata) tested for phytostabilization. Experiments were performed with an alkaline soil naturally rich in As. Composition of the soil solution of planted and unplanted pots was monitored every 15 days for 90 days, with a focus on the evolution of As concentrations in solution and in the non-specifically bound (i.e. easily exchangeable) fraction. The four species similarly increased As concentration in solution, but decreased As concentration in the non-specifically bound fraction. The major part (60%) of As desorbed from the non-specifically bound fraction in planted pots was likely redistributed on the less available fractions of As on the solid phase. A second part (35%) of desorbed As was taken up by plants. The minor part (5%) of desorbed As supplied As increase in solution. To conclude, plants induced a substantial redistribution of As on the less available fractions in the rhizosphere, as expected in phytostabilization strategies. Plants however concomitantly increased As concentration in the rhizosphere solution which may contribute to As transfer through plant uptake and leaching.

(13) CO2 /(12) CO2 exchange fluxes in a clamp-on leaf cuvette: disentangling artefacts and flux components.

Leaks and isotopic disequilibria represent potential errors and artefacts during combined measurements of gas exchange and carbon isotope discrimination (Δ). This paper presents new protocols to quantify, minimize, and correct such phenomena. We performed experiments with gradients of CO2 concentration (up to ±250 µmol mol(-1) ) and δ(13) CCO2 (34‰), between a clamp-on leaf cuvette (LI-6400) and surrounding air, to assess (1) leak coefficients for CO2 , (12) CO2 , and (13) CO2 with the empty cuvette and with intact leaves of Holcus lanatus (C3 ) or Sorghum bicolor (C4 ) in the cuvette; and (2) isotopic disequilibria between net photosynthesis and dark respiration in light. Leak coefficients were virtually identical for (12) CO2 and (13) CO2 , but ∼8 times higher with leaves in the cuvette. Leaks generated errors on Δ up to 6‰ for H. lanatus and 2‰ for S. bicolor in full light; isotopic disequilibria produced similar variation of Δ. Leak errors in Δ in darkness were much larger due to small biological : leak flux ratios. Leak artefacts were fully corrected with leak coefficients determined on the same leaves as Δ measurements. Analysis of isotopic disequilibria enabled partitioning of net photosynthesis and dark respiration, and indicated inhibitions of dark respiration in full light (H. lanatus: 14%, S. bicolor: 58%).

Warming differentially influences the effects of drought on stoichiometry and metabolomics in shoots and roots.

Plants in natural environments are increasingly being subjected to a combination of abiotic stresses, such as drought and warming, in many regions. The effects of each stress and the combination of stresses on the functioning of shoots and roots have been studied extensively, but little is known about the simultaneous metabolome responses of the different organs of the plant to different stresses acting at once. We studied the shift in metabolism and elemental composition of shoots and roots of two perennial grasses, Holcus lanatus and Alopecurus pratensis, in response to simultaneous drought and warming. These species responded differently to individual and simultaneous stresses. These responses were even opposite in roots and shoots. In plants exposed to simultaneous drought and warming, terpenes, catechin and indole acetic acid accumulated in shoots, whereas amino acids, quinic acid, nitrogenous bases, the osmoprotectants choline and glycine betaine, and elements involved in growth (nitrogen, phosphorus and potassium) accumulated in roots. Under drought, warming further increased the allocation of primary metabolic activity to roots and changed the composition of secondary metabolites in shoots. These results highlight the plasticity of plant metabolomes and stoichiometry, and the different complementary responses of shoots and roots to complex environmental conditions.

Influence of plant root exudates on the mobility of fuel volatile compounds in contaminated soils.

Vegetation and its associated microorganisms play an important role in the behaviour of soil contaminants. One of the most important elements is root exudation, since it can affect the mobility, and therefore, the bioavailability of soil contaminants. In this study, we evaluated the influence of root exudates on the mobility of fuel derived compounds in contaminated soils. Samples of humic acid, montmorillonite, and an A horizon from an alumi-umbric Cambisol were contaminated with volatile contaminants present in fuel: oxygenates (MTBE and ETBE) and monoaromatic compounds (benzene, toluene, ethylbenzene and xylene). Natural root exudates obtained from Holcus lanatus and Cytisus striatus and ten artificial exudates (components frequently found in natural exudates) were added to the samples, individually and as a mixture, to evaluate their effects on contaminant mobility. Fuel compounds were analyzed by headspace-gas chromatography-mass spectrometry. In general, the addition of natural and artificial exudates increased the mobility of all contaminants in humic acid. In A horizon and montmorillonite, natural or artificial exudates (as a mixture) decreased the contaminant mobility. However, artificial exudates individually had different effects: carboxylic components increased and phenolic components decreased the contaminant mobility. These results established a base for developing and improving phytoremediation processes of fuel-contaminated soils.

Trait-directed de novo population transcriptome dissects genetic regulation of a balanced polymorphism in phosphorus nutrition/arsenate tolerance in a wild grass, Holcus lanatus.

The aim of this study was to characterize the transcriptome of a balanced polymorphism, under the regulation of a single gene, for phosphate fertilizer responsiveness/arsenate tolerance in wild grass Holcus lanatus genotypes screened from the same habitat. De novo transcriptome sequencing, RNAseq (RNA sequencing) and single nucleotide polymorphism (SNP) calling were conducted on RNA extracted from H. lanatus. Roche 454 sequencing data were assembled into c. 22,000 isotigs, and paired-end Illumina reads for phosphorus-starved (P-) and phosphorus-treated (P+) genovars of tolerant (T) and nontolerant (N) phenotypes were mapped to this reference transcriptome. Heatmaps of the gene expression data showed strong clustering of each P+/P- treated genovar, as well as clustering by N/T phenotype. Statistical analysis identified 87 isotigs to be significantly differentially expressed between N and T phenotypes and 258 between P+ and P- treated plants. SNPs and transcript expression that systematically differed between N and T phenotypes had regulatory function, namely proteases, kinases and ribonuclear RNA-binding protein and transposable elements. A single gene for arsenate tolerance led to distinct phenotype transcriptomes and SNP profiles, with large differences in upstream post-translational and post-transcriptional regulatory genes rather than in genes directly involved in P nutrition transport and metabolism per se.

Influence of soil properties and phosphate addition on arsenic uptake from polluted soils by velvetgrass (Holcus lanatus).

Four kinds of soil material were used in a pot experiment with velvetgrass (Holcus lanatus). Two unpolluted soils: sand (S) and loam (L) were spiked with sodium arsenite (As II) and arsenate (As V), to obtain total arsenic (As) concentrations of 500 mg As kg(-1). Two other soils (ZS I, ZS III), containing 3320 and 5350 mg As kg(-1), were collected from Zloty Stok where gold and arsenic ores were mined and processed for several centuries. The effects of phosphate addition on plants growth and As uptake were investigated. Phosphate was applied to soils in the form of NH4H2PO4 at the rate 0.2 g P/kg. Average concentrations of arsenic in the shoots of velvetgrass grown in spiked soils S and L without P amendment were in the range 18-210 mg As kg(-1) d.wt., whereas those in plants grown on ZS I and ZS II soils were considerably lower, and varied in the range 11-52 mg As kg(-1) d.wt. The addition of phosphate caused a significant increase in plant biomass and therefore the total amounts of As taken up by plants, however, the differences in As concentrations in the shoots of velvetgrass amended and non-amended with phosphate were not statistically significant.

Arsenic extractability and uptake by velvetgrass Holcus lanatus and ryegrass Lolium perenne in variously treated soils polluted by tailing spills.

Phytostabilization should be considered as an appropriate phytoremediation technique to restore the area affected by tailing spills in Zloty Stok, where arsenic ores were mined and processed for several centuries. The study aimed to compare the suitability of velvetgrass (Holcus lanatus L.) and ryegrass (Lolium perenne L.) for development of plant cover in that area. Various treatments commonly applied to support phytostabilization were examined. A pot experiment was carried out to assess the effects of soil amendment with phosphate (P), sewage sludge (SS) and iron salts (Fe) on arsenic extractability and its uptake by grass. Four kinds of soil material, containing 356-5350 mg kg(-1) As, were examined. Velvetgrass proved to be more resistant than ryegrass to the toxicity of soil arsenic. Ammonium sulphate extractability of As in soils correlated well with As concentrations in the biomass of both grass species. In three of four tested soils, application of Fe failed to decrease As extractability and to reduce its concentrations in the aboveground parts of grasses. Application of P and SS resulted in increased As solubility in soils, but their effects on plant biomass and As uptake were ambiguous. SS had a strong beneficial influence on the growth of velvetgrass, while such an effect was not observed for ryegrass.

Species-specific effects of live roots and shoot litter on soil decomposer abundances do not forecast plant litter-nitrogen uptake.

Plant species produce litter of varying quality and differ in the quality and quantity of compounds they release from live roots, which both can induce different decomposer growth in the soil. To test whether differences in decomposer growth can forecast the amount of N species acquire from plant litter, as suggested by theory, we grew individuals of three grassland plants-Holcus lanatus, Plantago lanceolata and Lotus corniculatus-in soils into which (15)N-labelled litter of either Holcus, Plantago or Lotus was added. We measured the effects of live roots and litter of each species on soil microbes and their protozoan and nematode feeders, and to link decomposer growth and plant nutrient uptake, we measured the amount of N taken up by plants from the added litter. We hypothesised that those species that induce the highest growth of microbes, and especially that of microbial feeders, will also take up the highest amount of N from the litter. We found, however, that although numbers of bacterial-feeding Protozoa and nematodes were on average lower after addition of Holcus than Plantago or Lotus litter, N uptake was higher from Holcus litter. Further, although the effects on Protozoa and bacterial- and fungal-feeding nematodes did not differ between the live plants, litter-N uptake differed, with Holcus being the most efficient compared to Plantago and Lotus. Hence, although microbes and their feeders unquestionably control N mineralization in the soil, and their growth differs among plant species, these differences cannot predict differences in litter-N uptake among plant species. A likely reason is that for nutrient uptake, other species-specific plant traits, such as litter chemistry, root proliferation ability and competitiveness for soil N, override in significance the species-specific ability of plants to induce decomposer growth.

Arsenite efflux is not enhanced in the arsenate-tolerant phenotype of Holcus lanatus.

Arsenate tolerance in Holcus lanatus is achieved mainly through suppressed arsenate uptake. We recently showed that plant roots can rapidly efflux arsenite to the external medium. Here, we tested whether arsenite efflux is a component of the adaptive arsenate tolerance in H. lanatus. Tolerant and nontolerant phenotypes were exposed to different arsenate concentrations with or without phosphate for 24 h, and arsenic (As) speciation was determined in nutrient solutions, roots and xylem sap. At the same arsenate exposure concentration, the nontolerant phenotype took up more arsenate and effluxed more arsenite than the tolerant phenotype. However, arsenite efflux was proportional to arsenate uptake and was not enhanced in the tolerant phenotype. Within 2-24 h, most (80-100%) of the arsenate taken up was effluxed to the medium as arsenite. About 86-95% of the As in the roots and majority of the As in xylem sap (c. 66%) was present as arsenite, and there were no significant differences between phenotypes. Arsenite efflux is not adaptively enhanced in the tolerant phenotype H. lanatus, but it could be a basal tolerance mechanism to greatly decrease cellular As burden in both phenotypes. Tolerant and nontolerant phenotypes had a similar capacity to reduce arsenate in roots.

Nutrient availability and atmospheric CO2 partial pressure modulate the effects of nutrient heterogeneity on the size structure of populations in grassland species.

BACKGROUND AND AIMS: Size-asymmetric competition occurs when larger plants have a disproportionate advantage in competition with smaller plants. It has been hypothesized that nutrient heterogeneity may promote it. Experiments testing this hypothesis are inconclusive, and in most cases have evaluated the effects of nutrient heterogeneity separately from other environmental factors. The aim of this study was to test, using populations of Lolium perenne, Plantago lanceolata and Holcus lanatus, two hypotheses: (a) nutrient heterogeneity promotes size-asymmetric competition; and (b) nutrient heterogeneity interacts with both atmospheric CO2 partial pressure (P(CO2)) and nutrient availability to determine the magnitude of this response. METHODS: Microcosms consisting of monocultures of the three species were grown for 90 d in a factorial experiment with the following treatments: P(CO2) (37.5 and 70 Pa) and nutrient availability (NA; 40 and 120 mg of N added as organic material) combined with different spatial distribution of the organic material (NH; homogeneous and heterogeneous). Differences in the size of individual plants within populations (size inequality) were quantified using the coefficient of variation of individual above-ground biomass and the combined biomass of the two largest individuals in each microcosm. Increases in size inequality were associated with size-asymmetric competition. KEY RESULTS: Size inequality increased when the nutrients were heterogeneously supplied in the three species. The effects of NH on this response were more pronounced under high nutrient supply in both Plantago and Holcus (significant NA x NH interactions) and under elevated P(CO2) in Plantago (significant P(CO2) x NA x NH interaction). No significant two- and three-way interactions were found for Lolium. CONCLUSIONS: Our first hypothesis was supported by our results, as nutrient heterogeneity promoted size-asymmetric competition in the three species evaluated. Nutrient supply and P(CO2) modified the magnitude of this effect in Plantago and Holcus, but not in Lolium. Thus, our second hypothesis was partially supported.

The nature of arsenic-phytochelatin complexes in Holcus lanatus and Pteris cretica.

We have developed a method to extract and separate phytochelatins (PCs)-metal(loid) complexes using parallel metal(loid)-specific (inductively coupled plasma-mass spectrometry) and organic-specific (electrospray ionization-mass spectrometry) detection systems-and use it here to ascertain the nature of arsenic (As)-PC complexes in plant extracts. This study is the first unequivocal report, to our knowledge, of PC complex coordination chemistry in plant extracts for any metal or metalloid ion. The As-tolerant grass Holcus lanatus and the As hyperaccumulator Pteris cretica were used as model plants. In an in vitro experiment using a mixture of reduced glutathione (GS), PC(2), and PC(3), As preferred the formation of the arsenite [As((III))]-PC(3) complex over GS-As((III))-PC(2), As((III))-(GS)(3), As((III))-PC(2), or As((III))-(PC(2))(2) (GS: glutathione bound to arsenic via sulphur of cysteine). In H. lanatus, the As((III))-PC(3) complex was the dominant complex, although reduced glutathione, PC(2), and PC(3) were found in the extract. P. cretica only synthesizes PC(2) and forms dominantly the GS-As((III))-PC(2) complex. This is the first evidence, to our knowledge, for the existence of mixed glutathione-PC-metal(loid) complexes in plant tissues or in vitro. In both plant species, As is dominantly in non-bound inorganic forms, with 13% being present in PC complexes for H. lanatus and 1% in P. cretica.

The distribution of arsenate and arsenite in shoots and roots of Holcus lanatus is influenced by arsenic tolerance and arsenate and phosphate supply.

The recent discovery that phytochelatins are important for arsenic (As) detoxification in terrestrial plants results in the necessity to understand As speciation and metabolism in plant material. A hydroponic study was therefore conducted to examine the effects of different levels of phosphate and arsenate [As(V)] on As speciation and distribution in tolerant and non-tolerant clones of Holcus lanatus. Speciation of As in tissue (using high-performance liquid chromatography-inductively coupled plasma mass spectrometry) revealed that the predominant species present were the inorganic As species (As(V) and arsenite [As(III)]), although small levels (<1%) of organic As species (dimethylarsinic acid and monomethylarsonic acid) were detected in shoot material. In roots, the proportion of total As present as As(III) generally increased with increasing levels of As(V) in the nutrient solution, whereas in shoots, the proportion of total As present as As(III) generally decreased with increasing levels of As(V). H. lanatus plants growing in the high-phosphorus (P) (100 micro M) solution contained a higher proportion of As(V) (with regard to total As) in both roots and shoots than plants supplied with low P (10 micro M); in addition, tolerant clones generally contained a higher proportion of As(V) with regard to total As than non-tolerant clones. The study further revealed that As(V) can be reduced to As(III) in both roots and shoots. Although the reduction capacity was limited, the reduction was closely regulated by As influx for all treatments. The results therefore provide a new understanding about As metabolism in H. lanatus.