Juan Sobrino-Plata

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.

Contribution of phytochelatins to cadmium tolerance in peanut plants

Cadmium (Cd) is a well known heavy metal considered as one of the most toxic metals on Earth, affecting all viable cells that are exposed even at low concentration. It is introduced to agricultural soils mainly by phosphate fertilizers and causes many toxic symptoms in cells. Phytochelatins (PCs) are non-protein thiols which are involved in oxidative stress protection and are strongly induced by Cd. In this work, we analyzed metal toxicity as well as PCs implication on protection of peanut plants exposed to Cd. Results showed that Cd exposure induced a reduction of peanut growth and produced changes in the histological structure with a deposit of unknown material on the epidermal and endodermal cells. When plants were exposed to 10 μM Cd, no modification of chlorophyll, lipid peroxides, carbonyl groups, or hydrogen peroxide (H₂O₂) content was observed. At this concentration, peanut leaves and roots glutathione (GSH) content decreased. However, peanut roots were able to synthesize different types of PCs (PC2, PC3, PC4). In conclusion, PC synthesis could prevent metal disturbance on cellular redox balance, avoiding oxidative damage to macromolecules.

Specific stress responses to cadmium, arsenic and mercury appear in the metallophyte Silene vulgaris when grown hydroponically

The tolerance of the metallophyte Silene vulgaris, a plant suitable for the phytostabilisation of metal(loid)-contaminated soils, to arsenic (As), mercury (Hg) and cadmium (Cd) was evaluated in a semi-hydroponic culture system under controlled environmental conditions. The appearance of oxidative stress, alteration of photochemical processes and modification of biothiol content were studied as physiological parameters of metal(loid) stress in plants treated with 0, 6 and 30 μM (As, Hg or Cd) for 7 days. In spite of the metal(loid) excluder behaviour of S. vulgaris, Cd was translocated to the aerial part of the plant at a higher rate than Hg or As. The major toxic effects were observed in roots, where lipid peroxidation was increased in a dose-dependent manner. Redox enzymes such as glutathione reductase (GR) were severely inhibited by Hg, whereas GR was overexpressed. The accumulation of Cd produced a remarkable production of phytochelatins (PCs) in roots, whereas Hg and As led to modest PCs synthesis. There was a severe loss of chlorophyll content in Cd-treated plants, accompanied with a significant decrease in photosystem II efficiency (ΦPSII) and photochemical quenching (qP). Similar negative effects were observed in Hg- and As-exposed plants, but to a lesser degree. The exposure to the highest dose of each toxic element (30 μM) caused depletion of the light harvesting complex b1 protein. In conclusion, specific stress signatures to each metal(loid) were observed, with As being the least toxic element, suggesting that different mechanisms of tolerance were exerted. These results could be applied in future experiments to select tolerant ecotypes to optimize the phytostabilisation of metal(loid) multipolluted soils.