Sandra Carrasco-Gil

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.

Mercury localization and speciation in plants grown hydroponically or in a natural environment.

Better understanding of mercury (Hg) accumulation, distribution, and speciation in plants is required to evaluate potential risks for the environment and to optimize phytostabilization strategies for Hg-contaminated soils. The behavior of Hg in alfalfa (Medicago sativa) plants grown under controlled conditions in a hydroponic system (30 μM HgCl2) was compared with that of naturally occurring Horehound (Marrubium vulgare) plants collected from a mining soil polluted with Hg (Almadenejos, Spain) to characterize common mechanisms of tolerance. Synchrotron X-ray Fluorescence microprobe (μ-SXRF) showed that Hg accumulated at the root apex of alfalfa and was distributed through the vascular system to the leaves. Transmission electron microscopy (TEM) implied association of Hg with cell walls, accompanied by their structural changes, in alfalfa roots. Extended X-ray absorption fine structure (EXAFS) determined that Hg was principally bound to biothiols and/or proteins in M. sativa roots, stems, and leaves. However, the major fraction of Hg detected in M. vulgare plants consisted of mineral species, possibly associated with soil components. Interestingly, the fraction of Hg bound to biothiols/proteins (i.e., metabolically processed Hg) in leaves of both plants (alfalfa and M. vulgare) was similar, in spite of the big difference in Hg accumulation in roots, suggesting that some tolerance mechanisms might be shared.

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.

Using Perls Staining to Trace the Iron Uptake Pathway in Leaves of a Prunus Rootstock Treated with Iron Foliar Fertilizers

The aim of this study was to trace the Fe uptake pathway in leaves of Prunus rootstock (GF 677; Prunus dulcis × Prunus persica) plants treated with foliar Fe compounds using the Perls blue method, which detects labile Fe pools. Young expanded leaves of Fe-deficient plants grown in nutrient solution were treated with Fe-compounds using a brush. Iron compounds used were the ferrous salt FeSO4, the ferric salts Fe2(SO4)3 and FeCl3, and the chelate Fe(III)-EDTA, all of them at concentrations of 9 mM Fe. Leaf Fe concentration increases were measured at 30, 60, 90 min, and 24 h, and 70 μm-thick leaf transversal sections were obtained with a vibrating microtome and stained with Perls blue. In vitro results show that the Perls blue method is a good tool to trace the Fe uptake pathway in leaves when using Fe salts, but is not sensitive enough when using synthetic Fe(III)-chelates such as Fe(III)-EDTA and Fe(III)-IDHA. Foliar Fe fertilization increased leaf Fe concentrations with all Fe compounds used, with inorganic Fe salts causing larger leaf Fe concentration increases than Fe(III)-EDTA. Results show that Perls blue stain appeared within 30 min in the stomatal areas, indicating that Fe applied as inorganic salts was taken up rapidly via stomata. In the case of using FeSO4 a progression of the stain was seen with time toward vascular areas in the leaf blade and the central vein, whereas in the case of Fe(III) salts the stain mainly remained in the stomatal areas. Perls stain was never observed in the mesophyll areas, possibly due to the low concentration of labile Fe pools.

Effect of several commercial seaweed extracts in the mitigation of iron chlorosis of tomato plants (Solanum lycopersicum L.)

Commercial seaweed extracts (SWEs) have been applied in agriculture for ameliorating biotic and abiotic stress in plants. However, the mechanisms of action of these extracts are only partially known. Most of the research work with SWEs has focused on abiotic stresses such as drought, salinity or high temperatures, but little is known about SWE effects on plants with nutrient imbalances. Therefore, the goal of this study was to investigate the effects of several commercial SWEs (based on Ascophyllum nodosum and Durvillea potatorum) in the mitigation of iron chlorosis of stressed tomato plants. Tomato plants were grown in a hydroponic system initially with Fe, and then Fe was removed from the nutrient solution. SWEs were applied twice, first during the growth period (+ Fe) and second at the beginning of Fe deficiency (− Fe), following the recommended doses of manufacturers. Some of SWE treatments activated the antioxidant system in Fe-deficient tomato plants increasing SOD and CAT activity. However, SWEs application did not produce positive effects on biomass, chlorophyll content, activation of Fe acquisition strategies and Fe uptake with respect to the untreated control.