Luis E. Hernández

Alterations in the mineral nutrition of pea seedlings exposed to cadmium

Abstract A study was made of the effects of different levels of cadmium (Cd) in the nutrient solution (0.0, 1.5, and 6.0 mg/L Cd) on the assimilation of nitrate (NO3 ‐) and the uptake and distribution of cationic nutrients in pea (Pisum sativum cv. Argona) seedlings. Cadmium treatment resulted in an accumulation of NO3 ‐ in the root, indicating an alteration in NO3 ‐ translocation to the shoot. This was related to a decrease in the nitrate reductase (NR) activity in the shoot, severely inhibiting NO3 ‐ assimilation, and associated to a reduction in fresh tissue weight and in their relative water content. The concentration of potassium (K) decreased in both root and shoot, but its relative distribution between those tissues was not affected by the presence of Cd. Among other cationic nutrients, manganese (Mn) was the most affected, its concentration constantly decreased concomitantly with the increase in Cd supply. The distribution of Mn between shoot and root revealed that more was accumulated in the shoo...

Association of Phosphatidylinositol 3-Kinase with Nuclear Transcription Sites in Higher Plants

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti–bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Influence of cadmium on the uptake, tissue accumulation and subcellular distribution of manganese in pea seedlings

Pea (Pisum sativum L. cv. Argona) plants were challenged with Cd in a pure hydroponic system to study the effects on the uptake, tissue accumulation and subcellular distribution of Mn and Fe. This subcellular partitioning was compared to that of maize (Zea mays L. cv. Paolo) plants. In a long-term exposure experiment, Cd was supplied continuously with 0.0 (control), 10 or 50 μM Cd for 10 days. In a short-term treatment, pea plants were given 50 μM Cd for 72 h; this was followed by removing Cd from the nutrient solution and investigating the changes in the plants during the period of recovery from Cd toxicity for 96 h. The Relative Growth Increment (RGI) showed that plants treated with 10 μM Cd suffered moderate stress (70% of the control), whereas those given 50 μM experienced an strong phytotoxic effect (ca. 30%). In both experiments, Cd accumulated mostly in the roots (90% of the total plant concentration) and almost completely inhibited the uptake of Mn. Only when Cd was removed from the nutrient solution Mn uptake resumed. The relative distribution (%) of Mn between shoots and roots revealed that more Mn was found in shoots as Cd concentration increased in the nutrient solution (from 20% to ca. 50% in Cd-treated plants). Iron concentration and uptake showed less correlation with the Cd treatments. The exposure of pea plants to 10 μM and 50 μM Cd caused the severe depletion of Mn in all root subcellular fractions of pea and maize (Zea mays) plants. However, the Mn relative content (%) between subcellular fractions increased in the cell-wall containing fraction, but decreased in the soluble fraction. This effect was also observed in maize plants.

Differential alterations of antioxidant defenses as bioindicators of mercury and cadmium toxicity in alfalfa.

Several physiological parameters related to oxidative stress, which is a characteristic of plants exposed to toxic metals, were studied in 3-week-old alfalfa plants treated with cadmium (Cd) or mercury (Hg) at doses of 0, 3, 10 and 30 microM for 7d. The concentrations of biothiols, glutathione (GSH), homoglutathione (hGSH) and phytochelatins (PCs) increased dramatically in metals-treated plants, in particular in the presence of Cd. This was accompanied by a remarkable up-regulation of gamma-glutamyl cysteine synthetase gene, probably in response to the higher demand for GSH|hGSH needed for PC synthesis. The presence of metals enhanced lipid peroxidation in shoots, while chlorophyll content declined in a concentration dependent manner. Ascorbate peroxidase (APX) activity increased moderately in roots of Cd-exposed plants, and a new basic root peroxidase isoform was found in both Cd- and Hg-treated plants. Glutathione reductase (GR) activity was enhanced in shoots of plants exposed to Cd and Hg. However, this enzymatic activity showed a metal dependent response in roots, and was enhanced in Cd-treated plants but was severely inhibited in roots of plants treated with Hg. Inhibition of GR by Hg was confirmed in vitro by incubating a commercially available GR and control shoot extracts with several doses of Hg and Cd. Ascorbate concentrations were elevated with treatments of 3 microM Hg, 10 microM Cd and 30 microM Cd, indicating that this compound is necessary for redox cellular homeostasis. The different responses observed with Cd and Hg treatments might be the basis for specific stress bioindicators.

Contribution of glutathione to the control of cellular redox homeostasis under toxic metal and metalloid stress

The accumulation of toxic metals and metalloids, such as cadmium (Cd), mercury (Hg), or arsenic (As), as a consequence of various anthropogenic activities, poses a serious threat to the environment and human health. The ability of plants to take up mineral nutrients from the soil can be exploited to develop phytoremediation technologies able to alleviate the negative impact of toxic elements in terrestrial ecosystems. However, we must select plant species or populations capable of tolerating exposure to hazardous elements. The tolerance of plant cells to toxic elements is highly dependent on glutathione (GSH) metabolism. GSH is a biothiol tripeptide that plays a fundamental dual role: first, as an antioxidant to mitigate the redox imbalance caused by toxic metal(loid) accumulation, and second as a precursor of phytochelatins (PCs), ligand peptides that limit the free ion cellular concentration of those pollutants. The sulphur assimilation pathway, synthesis of GSH, and production of PCs are tightly regulated in order to alleviate the phytotoxicity of different hazardous elements, which might induce specific stress signatures. This review provides an update on mechanisms of tolerance that depend on biothiols in plant cells exposed to toxic elements, with a particular emphasis on the Hg-triggered responses, and considering the contribution of hormones to their regulation.

Complexation of Hg with phytochelatins is important for plant Hg tolerance

Three-week-old alfalfa (Medicago sativa), barley (Hordeum vulgare) and maize (Zea mays) were exposed for 7 d to 30 µm of mercury (HgCl(2) ) to characterize the Hg speciation in root, with no symptoms of being poisoned. The largest pool (99%) was associated with the particulate fraction, whereas the soluble fraction (SF) accounted for a minor proportion (<1%). Liquid chromatography coupled with electro-spray/time of flight mass spectrometry showed that Hg was bound to an array of phytochelatins (PCs) in root SF, which was particularly varied in alfalfa (eight ligands and five stoichiometries), a species that also accumulated homophytochelatins. Spatial localization of Hg in alfalfa roots by microprobe synchrotron X-ray fluorescence spectroscopy showed that most of the Hg co-localized with sulphur in the vascular cylinder. Extended X-ray Absorption Fine Structure (EXAFS) fingerprint fitting revealed that Hg was bound in vivo to organic-S compounds, i.e. biomolecules containing cysteine. Albeit a minor proportion of total Hg, Hg-PCs complexes in the SF might be important for tolerance to Hg, as was found with Arabidopsis thaliana mutants cad2-1 (with low glutathione content) and cad1-3 (unable to synthesize PCs) in comparison with wild type plants. Interestingly, high-performance liquid chromatography-electrospray ionization-time of flight analysis showed that none of these mutants accumulated Hg-biothiol complexes.

WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis

This work shows that plants respond to arsenate by immediately freezing its uptake through the action of a transcriptional repressor of phosphate transporters and that the same transcription factor influences transposon expression in response to arsenate. Plants therefore have an arsenate perception mechanism that controls arsenate uptake and transposon expression, providing an integrated strategy for arsenate tolerance and genome stability. Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing.

Differential response of Arabidopsis leaves and roots to cadmium: glutathione-related chelating capacity vs antioxidant capacity.

This study aims to uncover the spatiotemporal involvement of glutathione (GSH) in two major mechanisms of cadmium (Cd)-induced detoxification (i.e. chelation and antioxidative defence). A kinetic study was conducted on hydroponically grown Arabidopsis thaliana (L. Heyhn) to gain insight into the early events after exposure to Cd. Cadmium detoxification was investigated at different levels, including gene transcripts, enzyme activities and metabolite content. Data indicate a time-dependent response both within roots and between plant organs. Early on in roots, GSH was preferentially allocated to phytochelatin (PC) synthesis destined for Cd chelation. This led to decreased GSH levels, without alternative pathways activated to complement GSHs antioxidative functions. After one day however, multiple antioxidative pathways increased including superoxide dismutase (SOD), ascorbate (AsA) and catalase (CAT) to ensure efficient neutralization of Cd-induced reactive oxygen species (ROS). As a consequence of Cd retention and detoxification in roots, a delayed response occurred in leaves. Together with high leaf thiol contents and possibly signalling responses from the roots, the leaves were protected, allowing them sufficient time to activate their defence mechanisms.

Glutamate measured by 6-s resolution brain microdialysis: capillary electrophoretic and laser-induced fluorescence detection application

In the present experiment the combination of brain microdialysis and CZE-LIFD permitted the measurement of glutamate in 100 nl microdialysis samples collected every 5 or 6 s. Samples were collected every 6 s, in rats anesthetized with two different anesthetic agents (ketamine and sodium thiopental). A microdialysis probe was inserted in the cortex of an anesthetized rat in the territory irrigated by the middle cerebral artery. The artery was clamped for 30 s and then released. The samples were derivatized with fluorescein isothiocyanate I (FITC) by means of a continuous-flow reactor, collected and injected into a home-made CZE-LIFD instrument. Glutamate decreased immediately after clamping the artery in ketamine anesthetized rats and increased 1 min after the onset of the ischemia in sodium thiopental anesthetized rats. In another experiment a 60 mM KCl solution was injected through a microdialysis probe inserted in the hippocampus of an anesthetized rat. In the first 5 s after the KCl solution reached the tissue, glutamate increased but gamma-aminobutytic acid and glutamine did not. The experiments show that time resolution of brain microdialysis can be reduced to a few seconds if the analytical technique is the proper one.

Toxoplasma gondii infection lower anxiety as measured in the plus-maze and social interaction tests in rats: A behavioral analysis

It has been suggested that the parasite Toxoplasma gondii reduces the fear of rodents toward their feline predators, which may lead to an augmented rate of predation and multiplication of the parasite through an increased number of life cycles. To investigate whether T. gondii infection induces selective effects on behavior associated with anxiety, Wistar rats were inoculated i.p. with several doses of T. gondii tachyzoites and tested in two animal tests of anxiety. In the third week following inoculation, rats infected with 100 and 1000 tachyzoites increased plus-maze open arm exploration in a dose-related manner. However, no effect was detected in either social interaction levels or motor activity measures. In the seventh week after inoculation, rats infected with 100 and 1000 tachyzoites showed increased open arm exploration and social investigation without change on any motor activity measures. However, rats infected with a higher dose (1500 tachyzoites) showed a drop in locomotion. These data support the hypothesis that T. gondii impairs mechanism of warning as a function of reduced anxiety. The pattern of brain colonization by the parasite and the host immune response suggests that the predominant invasion to limbic areas works as a natural anxiolytic mechanism.