Ramón O. Carpena

Bioavailability of metals and As from acidified multicontaminated soils: Use of white lupin to validate several extraction methods

White lupin is an annual crop that has been used for phytostabilization of acidified multicontaminated (heavy metals and As) soils from the Aznalcóllar spill-affected area, Southern Spain. One of the most important factors for successful phytostabilization is monitoring the pollutant bioavailability in the soil. The aim of this work was to determine the best-suited method for assessing the bioavailability of heavy metals together with As in the Aznalcóllar spill-affected area, by means of a systematic comparison between different extraction methods (Ammonium bicarbonate-diethylenetriamine pentaacetic acid (AB-DTPA), CaCl2, NaNO3, BCR, (NH4)2SO4 and rhizo). Both AB-DTPA and the first step of the BCR method were found to be unsuitable for assessing the bioavailability of heavy metals and As to plants growing in acidic soils. However, CaCl2-extractable As, Cu, and Zn and NaNO3-extractable As and Zn were well correlated with their concentrations in plant organs. Rhizo and (NH4)2SO4, with the highest determination coefficients, were the most recommended simple extraction methods to assess the bioavailability of As, Cu, Fe, Mn, and Zn in acidified multicontaminated soils using white lupin as an excluder model plant.

Boron and calcium distribution in nitrogen-fixing pea plants.

In a glasshouse experiment, plants of Pisum sativum L. cv. Argona were grown hydroponically with different B and Ca levels, in order to elucidate a specific role for B and Ca on the N(2) fixation in this temperate legume. The treatments were as follows: control (9.3 µM B and 2 mM Ca), -B (without B and 2 mM Ca), -B+Ca (without B and 3.6 mM Ca), +Ca (9.3 µM B and 3.6 mM Ca), -Ca (9.3 µM B and 0.4 mM Ca) and -Ca+B (46.5 µM B and 0.4 mM Ca). The supply of -Ca and +Ca did not affect nitrogenase activity, but the weight of old shoots and total N content increased with the Ca treatment. No symptoms of B deficiency were detected in the plants of the -B and -B+Ca treatments, apart from weight reduction in young shoots and lower nitrogenase activity. The B concentration decreased in young shoots and roots of plants grown without B (-B), but there was a sharper decrease in the roots of -B+Ca plants and the levels of B in the young shoots were similar to the control levels. The B concentration in -Ca plants was reduced in the old shoot and in the root, while plant weight and N content increased in -Ca+B plants. The cell wall and total B concentrations in the nodules were 4-fold compared with those of the roots. By contrast, the Ca root wall was 2.5 times higher than the nodule levels although total pectin was higher in the nodule than in the root. Finally, the results obtained showed that a high supply of Ca could induce B mobilisation from root to shoot. On the other hand, the high B requirement found in pea plant nodules may contribute to explain the low nitrogenase activity detected under -B conditions.

Comparative analysis of the contribution of phytochelatins to cadmium and arsenic tolerance in soybean and white lupin

The biosynthesis of phytochelatins (PCs) plays a crucial role in the detoxification and homeostasis of heavy metals and metalloids in plants. However, in an increasing number of plant species metal(loid) tolerance is not well correlated with the accumulation of PCs: tolerant ecotypes frequently contain lower levels of PCs than non-tolerant ecotypes. In this study we have compared the responses of soybean (Glycine max L. cv. Resnik) and white lupin (Lupinus albus L. cv. Marta) to cadmium and arsenate in order to assess the role of homophytochelatins (hPCs) in the tolerance of soybean to these toxic elements. Soybean plants treated with Cd and As showed a high contribution of homo-glutathione (hGSH) to the pool of thiols in shoots in comparison to white lupin. Higher levels of hPCs in Cd-treated soybeans compared to PCs in lupins did not prevent growth inhibition. In contrast, the role of hPCs in the detoxification mechanism to arsenate in soybean seems to be clearer, showing higher thiol concentrations and lower growth reductions than those present in lupin plants.

Effects of cadmium on cork oak (Quercus suber L.) plants grown in hydroponics

Cork oak (Quercus suber L.) is an autochthonous tree species that is being used for reforestation in heavy-metal-contaminated areas in Spain. A hydroponics experiment was carried out to characterize the effects of Cd on several morphological and physiological parameters in this species, including shoot length, nutrient concentrations and allocation in different organs, leaf pigment concentrations, photosynthetic efficiency, root ferric chelate reductase (FCR) activity and organic acid concentrations in xylem sap. Four different Cd treatments were applied, adding Cd chelated with EDTA or as chloride salt at two different concentrations (10 and 50 µM Cd). After 1 month of Cd treatment, plant growth was significantly inhibited in all treatments. Results indicate that Cd accumulates in all organs 7- to 500-fold when compared with control plants. The highest Cd concentration was found in the 50 µM CdCl(2) treatment, which led to concentrations of ~30, 123 and 1153 µg Cd g(-1) dry weight in leaves, stems and roots, respectively. In the strongest Cd treatments the concentrations of P and Ca decreased in some plant parts, whereas the Mn leaf concentrations decreased with three of the four Cd treatments applied. The concentrations of chlorophyll and carotenoids on an area basis decreased, whereas the (zeaxanthin plus antheraxanthin)/(total violaxanthin cycle carotenoids) ratio and the non-photochemical quenching increased significantly in all Cd treatments. Cadmium treatments caused significant increases in the activity of the enzyme FCR in roots and in the concentrations of organic acids in xylem sap. Some of the physiological changes found support the fact that Cd induces a deficiency of Fe in cork oak, although the plant Fe concentrations were not reduced significantly. At higher concentrations the effects of Cd were more pronounced, and were more marked when Cd was in the free ion form than when present in the form of Cd-EDTA.

Evolution of arsenate toxicity in nodulated white lupine in a long-term culture.

White lupine is an As-resistant legume that is of interest for phytoremediation of As-contaminated soils. To achieve successful phytoremediation, monitoring of the nutritional status of the selected plant species during the entire culture cycle is required to maintain a plant cover with high biomass production. A long-term pot experiment was carried out with nodulated lupine grown on perlite with 10 and 100 microM As concentrations. The reproductive period (from 10 weeks) was the most sensitive phenologic stage of white lupine to long-term As exposure. The 10 microM As treatment increased the uptake and translocation of micronutrients, except for Cu, mainly at flowering with As levels in pods below the statutory limit (1 mg kg (-1) fresh weight). However, the 100 microM As treatment induced significant differences compared to the control. These findings confirm the relatively high resistance of white lupine to arsenate and support the use of this species in phytoremediation and/or revegetation of As-contaminated sites, with special attention on P and Cu nutrition at flowering.

Natural attenuation of residual heavy metal contamination in soils affected by the Aznalcóllar mine spill, SW Spain

Non-amended soils affected by pyritic sludge residues were monitored for 7 years to assess the long-term natural attenuation ability of these soils. The decrease in both the total concentration of elements (particularly As) and (NH(4))(2)SO(4)-extractable fractions of Mn, and Zn, below the maximum permissible levels indicate a successful natural ability to attenuate soil pollution. Soil acidification by pyrite oxidation and rainfall-enhanced leaching were the largest contributors to the reduction of metals of high (Mn, Cu, Zn and Cd) and low (Fe, Al, and As) availability. Periodic use of correlation and spatial distribution analysis was useful in monitoring elemental dispersion and soil property/element relationships.

Effect of 1-naphthaleneacetic acid on organic acid exudation by the roots of white lupin plants grown under phosphorus-deficient conditions.

The effect of NAA (1-naphthaleneacetic acid) on organic acid exudation in white lupin plants grown under phosphorus deficiency was investigated. Plants were sampled periodically for collecting of organic acids (citrate, malate, succinate), and also were used to study the effect on proton extrusion and release of Na(+), K(+), Ca(2+) and Mg(2+). The tissues were later processed to quantify the organic acids in tissues, the phosphorus content and the effects on plant biomass. The exogenous addition of NAA led to an increase in organic acid exudation, but this response was not proportional to the concentration of the dose applied, noticing the largest increments with NAA 10(-8)M. In contrast the increase in root weight was proportional to the dose applied, which shows that with higher doses the roots produced are not of proteoid type. Proton extrusion and the release of cations were related to the NAA dose, the first was proportional to the dose applied and the second inversely proportional. Regarding the analysis of tissues, the results of citrate and phosphorus content in shoots show that the overall status of these parts are the main responsible of the organic acids exuded. NAA served as an enhancer of the organic acid exudation that occurs under phosphorus deficient conditions, with a response that depends on the dose applied, not only in its magnitude, but also in the mechanism of action of the plant hormone.

Heavy Metal Perception in a Microscale Environment: A Model System Using High Doses of Pollutants

The characterization of the mechanisms of heavy metal detoxification has been undertaken through several experimental approaches, where high metal concentrations have been frequently used. A microscale hydroponic system was used to discriminate between the direct and indirect phytotoxic effects that may occur under heavy metal stress at short exposure times. Induction of oxidative stress and generation of stress signaling molecules are some of the physiological responses triggered soon after the exposure of plant cells to heavy metals, which might be part of stress perception mechanisms. The generation of reactive oxygen species, in particular H2O2, ethylene or jasmonate are envisaged as messengers in signaling pathways that may result ultimately in cell senescence and growth inhibition.

Influence of phosphorus supply on tomato plant nutrition

Abstract Four nutrient solutions with different P‐levels (0.2; 0.5; 2.0 and 4.0 mmol P/l) were studied in order to optimize P nutrition of the tomato plant. A N/P ratio decrease in sap and the leaf was observed when P supply increased. Moreover, excessive and deficient P levels in the nutrient solution resulted in a flavonol glycosides decrease in leaves, specially at flowering. Chlorophyll a/chlorophyll b ratios and chlorophyll a percentages, as compared with the total, were lower in the P‐0.2 and P‐0.5 treatments than in the P‐2.0 treatment.

Response of Lupinus albus to Pb–EDTA indicates relatively high tolerance

ABSTRACT White lupin plants were grown in hydroponics with 0, 90 and 180 µmol L−1 Pb(II) ethylenediaminetetraacetate complex for 30 days. Pb distribution (shoot/root ratio) was 0.34 and 0.46 for both Pb treatments. In the shoots, no decrease in biomass nor in photosynthetic pigment levels and no changes in the concentrations of malondialdehyde and glutathione were detected. In the roots, malondialdehyde increased by 20%, glutathione 2–3.6 times and phytochelatin concentrations 4–5 times. The high tolerance of white lupin makes it a valuable plant for phytoremediation of Pb-contaminated soil.