Juan J. Lucena

Cadmium uptake and subcellular distribution in plants of Lactuca sp. Cd–Mn interaction

Abstract Cd uptake and subcellular distribution in (Lactuca sativa cv. Grandes Lagos) was studied. In vivo and in vitro experiments were carried out to study Cd effect on Mn uptake and subcellular distribution. Lettuce plants were grown in hydroponics with nutrient solutions containing 0.1 and 1.0 mg Cd l−1 in a greenhouse (in vivo experiments). Also, Cd was directly infiltrated on lettuce leaves in in vitro studies (0.5 and 1 mg Cd l−1), to minimize absorption and transport processes. Root and shoots were sampled after 16 days of exposure to the Cd solutions. Cd, Mn and other micronutrients were assessed in the different parts of the plant sampled and in the subcellular fractions obtained after wet mineralization, using A.A. spectrophotometry. Results show that Cd was accumulated in leaves mainly in cell wall fraction (64%) and this accumulation was fairly independent of Cd level in nutrient solution. The lowest Cd concentration (12–14%) was found in chloroplasts for both Cd levels tested. The increase in Cd concentration in the external medium caused an increase in Mn uptake and translocation to the shoots of lettuce plants, in contrast to the behavior of the other essential micronutrients, and an increase in Mn content in the chloroplasts, suggesting an interaction between Cd and Mn at the chloroplast level.

Effects of bicarbonate, nitrate and other environmental factors on iron deficiency chlorosis. A review

Abstract The influence of several environmental factors on the occurrence of iron deficiency chlorosis on calcareous soils, also called bicarbonate induced chlorosis, is described. Bicarbonate in the soil solution is a strong pH buffer, mainly in the presence of calcium carbonate. Since bicarbonate is quite mobile, and CO2 diffusion is a slow process, the pH decrease in such soils after proton release by plants is small. Also, the ferric reductase activity of plant roots declines sharply at high pHs. The chemical Fe(III) reduction depends on the pe+pH; so, the lower the pH, the more favored is the formation of Fe(II) from the Fe(III) in the rizosphere. Nitrate can be acquired by the roots along with a proton cotransport that increase the pH outside the root plasmalemma, but a redox effect may be also important. In the rizosphere, the conditions for the reduction of nitrate (N(V)) to nitrite (N(III)) and ammonia (N(‐III)) are attained before than the conditions for the Fe(III) reduction from a solid phase. So, from the thermodynamic point of view, the electrons released by the plant can be taken directly by nitrate instead of Fe(III), or the Fe(II) formed can be reoxidized by the nitrate. Furthermore, if nitrate and nitrite reductions occurs in the rizosphere, microsite pH increases at the root surface. Since controversial data have been reported on the pH variations in the xylem sap in the presence of an excess of bicarbonate in the medium, a slower transport of iron does not appear to be the main reason for bicarbonate induced chlorosis. The “iron paradox” is well known (Römheld, 1987): sometimes, in soil culture, chlorotic leaves can have higher iron concentration than green ones. Some authors suggest that bicarbonate may affect the Fe distribution within the leaves. Apoplastic pH would control the entry of iron to the cytosol in leaves in a similar way than occurs in the root. Bicarbonate in the nutrient solution could act as a buffer in the leaf apoplast. Nitrate uptake in the meristem also produces a pH increase, with the same effect than the bicarbonate. Also, nitrate is an oxidant that could reduce the availability of the electrons for the Fe reduction for the membrane transport.

Effects of Cd and Pb in sugar beet plants grown in nutrient solution: induced Fe deficiency and growth inhibition

Effects of Cd and Pb toxicity were investigated in sugar beet (Beta vulgaris L.) grown in hydroponics under growth-chamber-controlled conditions. Chemical speciation calculations were used to estimate the chemical species in equilibrium. Cd, used as chloride salt or chelated to EDTA, decreased fresh and dry mass of both root and shoot, and increased root / shoot ratios. Plants developed few brownish roots with short laterals. Cd decreased N, P, Mg, K, Mn, Cu and Zn uptake, and facilitated Ca uptake. Leaves of plants treated with 10 or 50 μM Cd-EDTA and 10 μM CdCl2 developed symptoms of Fe deficiency. These symptoms included decreased leaf chlorophyll (Chl) and carotenoid concentrations, increased carotenoid / Chl and Chl a/b ratios, de-epoxidation of violaxanthin cycle pigments, and decreased photosynthetic rates and PSII efficiency. Plants treated with 50 μM CdCl2, however, had decreased growth but did not show marked leaf Fe-deficiency symptoms. All Cd treatments increased Fe(III)-chelate reductase activity in root tips, although Fe concentrations in shoots were similar to those found in control plants. Pb chelated with EDTA induced visual symptoms only at concentrations of 2 mM. Leaves of Pb-treated plants remained green and their edges were rolled inwards. Pb increased root fresh and dry mass with no changes in shoot mass, therefore increasing the root / shoot ratio. Changes in plant nutrient concentrations with Pb were only minor, although leaf Cu levels approached critical deficiency levels. No symptoms of Fe deficiency were apparent in leaves. Root tips of Pb-treated plants, however, had increased Fe(III)-chelate reductase activities.

The pH Requirement for in Vivo Activity of the Iron-Deficiency-Induced "Turbo" Ferric Chelate Reductase (A Comparison of the Iron-Deficiency-Induced Iron Reductase Activities of Intact Plants and Isolated Plasma Membrane Fractions in Sugar Beet).

The characteristics of the Fe reduction mechanisms induced by Fe deficiency have been studied in intact plants of Beta vulgaris and in purified plasma membrane vesicles from the same plants. In Fe-deficient plants the in vivo Fe(III)-ethylenediaminetetraacetic complex [Fe(III)-EDTA] reductase activity increased over the control values 10 to 20 times when assayed at a pH of 6.0 or below (“turbo” reductase) but increased only 2 to 4 times when assayed at a pH of 6.5 or above. The Fe(III)-EDTA reductase activity of root plasma membrane preparations increased 2 and 3.5 times over the controls, irrespective of the assay pH. The Km for Fe(III)-EDTA of the in vivo ferric chelate reductase in Fe-deficient plants was approximately 510 and 240 [mu]M in the pH ranges 4.5 to 6.0 and 6.5 to 8.0, respectively. The Km for Fe(III)-EDTA of the ferric chelate reductase in intact control plants and in plasma membrane preparations isolated from Fe-deficient and control plants was approximately 200 to 240 [mu]M. Therefore, the turbo ferric chelate reductase activity of Fe-deficient plants at low pH appears to be different from the constitutive ferric chelate reductase.

Fe Chelates for Remediation of Fe Chlorosis in Strategy I Plants

Abstract Iron chlorosis is a mineral disorder due to low Fe in the soil solution and the impaired plant uptake mechanism. These effects increased with high pH and bicarbonate buffer. The solution to Fe chlorosis should be made by either improving the Fe uptake mechanism or increasing the amount of Fe in the soil solution. Among Fe fertilizers, only the most stable chelates (EDDHA and analogous) are able to maintain Fe in the soil solution and transport it to the plant root. In commercial products with the same chelating agent, the efficacy depends on the purity and the presence of subproducts with complexing activity, that can be determined by appropriate analytical methods such as HPLC. In commercial products declaring 6% as Fe‐EDDHA, purity varied from 0.5% to 3.5% before 1999, but in 2002 products ranging 3–5.4% chelated Fe are common in the Spanish market. Fe‐o,p‐EDDHA, as a synthesis by‐product with unknown efficacy, is present in all Fe‐EDDHA formulations. Commercial Fe‐EDDHMA products also contain methyl positional isomers. Fe‐EDDHSA synthesis produces condensation products with similar chelating capacity to the Fe‐EDDHSA monomer that can account for more than 50% of the chelated iron in the commercial products. Chelates with different molecules should be compared for their efficacy considering firstly their ability to maintain Fe in solution and secondly their capacity to release iron to the roots. Accepting the turnover hypothesis, their efficacy is also dependent thirdly on the ability of the chelating agent to form the chelate using native iron from the soil. The 1st and 3rd points are related to the chemical stability of the chelate, while plants make better use of iron from the less stable chelates. Plant response is the ultimate evaluation method to compare commercial products with the same chelating agent or different chelates.

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.

Isocratic ion-pair high-performance liquid chromatographic method for the determination of various iron(III) chelates

Abstract The micronutrient iron, an essential element for plant growth, is usually added as fertilizer in chelated form. An isocratic ion-pair chromatographic method was developed to identify and determine the total amount of chelate in fertilizers. Iron(III) chelates containing ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, trans-1,2-cyclohexanediaminetetraacetic acid, ethylenediaminedi(o-hydroxyphenylacetic) acid (EDDHA), also known as N,N′-ethylenebis-2-(o-hydroxyphenyl)glycine, ethylenediaminedi(o-hydroxy-p-methylphenylacetic) acid, N,N′-bis-(2-hydroxybenzyl)ethylenediamine-N,N′-dipropionic acid and N,N-bis(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid were well separated by this method. The mobile phase contained 0.03 M tetrabutylammonium chloride and 30% acetonitrile at pH 6.0. The stationary phase was a LiChrospher RP-18 column, the injection volume was 20 μl and the flow-rate was 1.5 ml/min. For the iron(III)-EDDHA chelate, linear range studies showed that the method is capable of determining Fe concentrations between 0.5 and 150 μg/ml, which permits the determination of the concentrations found in commercial fertilizers. With this method, separation and identification of the iron(III) complexes were obtained with good resolution and selectivity, including the separation of the geometric isomers of the complexes, in 15 min.

Effectiveness of Ethylenediamine-N(o-hydroxyphenylacetic)-N′(p-hydroxyphenylacetic) acid (o,p-EDDHA) to Supply Iron to Plants

The Fe chelate o,p-EDDHA/Fe3+, in addition to o,o-EDDHA/Fe3+, was found recently to be a component of commercial EDDHA/Fe3+ chelates. The European Regulation on fertilisers has included o,p-EDDHA as an authorized chelating agent. The efficacy of o,o-EDDHA/Fe3+, o,p-EDDHA/Fe3+ and EDTA/Fe3+ chelates as Fe sources in plant nutrition was studied. Iron-chelate reductase (FC-R) in young cucumber plants (Cucumis sativus L.) roots reduced o,p-EDDHA/Fe3+ faster than o,o-EDDHA/Fe3+, EDTA/Fe3+ and a commercial source of EDDHA/Fe3+. The o,p-EDDHA/Fe3+ chelate was also more effective than the o,o-EDDHA/Fe3+ in decreasing the severity of Fe-deficiency chlorosis in leaves of young soybean (Glycine max L.) plants grown hydroponically. The o,p-EDDHA ligand was more effective in the short-term than the EDTA and o,o-EDDHA ligands at dissolving Fe from selected Fe minerals and soils. However, the ultimate quantity of dissolve Fe was greatest with the o,o-EDDHA ligand.

Synthetic Iron Chelates as Substrates of Root Ferric Chelate Reductase in Green Stressed Cucumber Plants

ABSTRACT This work studied the behavior of different iron (Fe)-chelates as substrates of ferric chelate reductase (FCR) and their ability as Fe suppliers for mildly chlorotic plants. FCR activity and Fe concentration in xylem sap were determined in green stressed cucumber plants with different stress levels using different synthetic chelates as substrates. Both reduction and Fe concentration in the xylem sap were higher for the less-stable Fe chelates, except for Fe-EDTA, which presented a relatively low Fe concentration in sap. It was concluded that a high stability of the chelate in the nutrient solution reduces the Fe reduction, but other factors, such as the complexation of the Fe(II) by the chelating agents, should be considered when the complete process of Fe uptake is studied. The use of both indexes together, i.e., FCR determination and xylem sap concentration, is useful for understanding the Fe uptake from different Fe chelates.

Chromatographic determination of commercial Fe(III) chelates of ethylenediaminetetraacetic acid, ethylenediaminedi(o-hydroxyphenylacetic) acid and ethylenediaminedi(o-hydroxy-p-methylphenylacetic) acid

Abstract The use of synthetic iron chelates is the most common and effective way to treat iron chlorosis in plants. Using an ion-pair HPLC method previously proposed by the authors, it was found that the older commercial products reached the percentage of Fe chelated indicated by the manufacturer, but in no case did the current products reach their nominal, or legal, composition. Moreover, the current products of Fe–ethylenediaminedi( o -hydroxyphenylacetic) acid (FeEDDHA) showed significant additional chromatographic peaks that, based on published synthesis pathways for these type of compounds, may correspond to para – para FeEDDHA or ortho – para FeEDDHA, sterically-hindered isomers of FeEDDHA which are of little or no value as an iron chelate for agricultural purposes.