Extraction of free amino acids from tomato leaves

Conditions for extraction of free amino acids from tomato leaves were examined. Two methods of sample preservation were also tested. Best results were obtained when samples were preserved by lyophilization and extracted by Soxhlet for 6 h at 40°C at a ratio of 1 g plant material/80 mL 80% (v/v) ethanol.     

Cellular damage induced by cadmium and mercury in Medicago sativa

Alfalfa (Medicago sativa) plantlets were exposed to Cd or Hg to study the kinetics of diverse stress indexes. In the so-called beaker-size hydroponic system, plantlets were grown in 30 microM of Cd or Hg for 7 d. Oxidative stress took place and increased over time, a linear response being observed with Cd but not with Hg. To improve the sensitivity of the stress assays used, a micro-assay system, in which seedlings were exposed for 24 h, was developed. Phytotoxicity of metals, quantified as growth inhibition, was observed well before there was any change in the non-protein thiol tissue concentration. When measured with conventional techniques, oxidative stress indexes did not show significant variation. To trace early and small plant responses to Cd and Hg, a microscopic analysis with novel fluorescent dyes, which had not yet been exploited to any significant extent for use in plants, was conducted. These fluorescent probes, which allowed minute cellular responses to 0, 3, 10, and 30 microM of both metals to be visualized in the roots of the alfalfa seedlings, were: (i) 2',7'-dichlorofluorescin diacetate that labels peroxides; (ii) monochlorobimane that stains reduced glutathione/homoglutathione (GSH/hGSH); and (iii) propidium iodide that marks nuclei of dead cells. Oxidative stress and cell death increased after exposure for 6-24 h to Cd and Hg, but labelling of GSH/hGSH decreased acutely. This diminution might be the result of direct interaction of GSH/hGSH with both Cd and Hg, as inferred from an in vitro conjugation assay. Therefore, both Cd and Hg not only compromised severely the cellular redox homeostasis, but also caused cell necrosis. In plants treated with 1 mM L-buthionine sulphoximine, a potent inhibitor of GSH/hGSH synthesis, only the oxidative stress symptoms appeared, indicating that the depletion of the GSH/hGSH pool was not sufficient to promote cell death, and that other phytotoxic mechanisms might be involved.     

Rapid alteration of cellular redox homeostasis upon exposure to cadmium and mercury in alfalfa seedlings

Here, the kinetics of oxidative stress responses of alfalfa (Medicago sativa) seedlings to cadmium (Cd) and mercury (Hg) (0, 3, 10 and 30 microm) exposure, expanding from a few minutes to 24 h, were studied. Intracellular oxidative stress was analysed using 2',7'-dichlorofluorescin diacetate and extracellular hydrogen peroxide (H(2)O(2)) production was studied with Amplex Red. Growth inhibition, concentrations of ascorbate, glutathione (GSH), homoglutathione (hGSH), Cd and Hg, ascorbate peroxidase (APX) activity, and expression of genes related to GSH metabolism were also determined. Both Cd and Hg increased cellular reactive oxygen species (ROS) production and extracellular H(2)O(2) formation, but in different ways. The increase was mild and slow with Cd, but more rapid and transient with Hg. Hg treatments also caused a higher cell death rate, significant oxidation of hGSH, as well as increased APX activity and transient overexpression of glutathione reductase 2, glutamylcysteinyl synthetase, and homoglutathione synthetase genes. However, Cd caused minor alterations. Hg accumulation was one order of magnitude higher than Cd accumulation. The different kinetics of early physiological responses in vivo to Cd and Hg might be relevant to the characterization of their mechanisms of toxicity. Thus, high accumulation of Hg might explain the metabolism poisoning observed in Hg-treated seedlings.     

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.     

Effects of cadmium on the uptake,distribution and assimilation of nitrate in Pisum sativum

The net uptake, distribution and assimilation of NO ₃ ^– were studied in pea plants subjected to either long-term continuous Cd treatment for 10 d (10 or 50 M Cd) or short-term treatment (72 h) with 50 M Cd. In the latter treatment, the effects of transferring the plants to a Cd-free nutrient solution for a 'recovery period' of 96 h were also studied. All these treatments were compared with 'controls', plants which received no Cd. In both experiments, the reduction in fresh weight was associated with a decrease in the content (%) of shoot and root water and in transpiration rate as Cd concentration increased. The concentration of ₃ ^– in the shoots and sap decreased dramatically and net ₃ ^– uptake was severely inhibited, effects associated with a loss of shoot nitrate reductase (NR) activity. In the short-term Cd treatment, net ₃ ^– uptake was almost completely inhibited after 24 h, but recovered after the transfer of plants to a Cd-free nutrient solution. Similarly, a dramatic decrease in the shoot NR activity was observed. The uptake, distribution and tissue partitioning of K was also studied, which is considered to be the major counterion of ₃ ^– . Potassium uptake was similarly affected by Cd, as inferred from the ratio ₃ ^– /K uptake, which was ca. 10. The ratio K/ ₃ ^– tissue content increased in the shoot concomitantly to Cd in both long-term and short-term metal supply. These parameters showed a tendency of K similar to that observed for ₃ ^– , although its relative tissue distribution was not affected by Cd.     

Purification of pea nodule symbiosomes using an aqueous polymer two-phase system

Symbiosomes were obtained from mature pea (Pisum sativum cv. Argona) root nodules infected with a Rhizobium leguminosarum strain (biov. viciae 3841) and purified using an aqueous polymer two-phase system (APS). The APS consists of a mixture of polymers, usually dextran T500 and poly(ethylene glycol) 3350, prepared as aqueous solutions on a weight per weight basis, where each fraction distributes according to their surface characteristics. Results of ATPase activity, cytochrome c oxidase activity, glucan synthase II activity, NAD(P)H-cytochrome c reductase activity, NO₃⁻-sensitive ATPase activity, transport of [¹⁴C]malate vs. [¹⁴C]glutamate and MAC 57 antigen analysis showed that the APS method provided intact symbiosomes with low bacteroid, plasma membrane, endoplasmic reticulum and/or mitochondria contamination. No complicated equipment is needed and the method was simple and fast, compared with other purification techniques.     

Distribution of cadmium in shoot and root tissues1

Maize and pea plants were treated with 0.0 (control), 0.01 or0.05 mM Cd in the growing medium for 11 d. Although the totalCd concentration was similar in shoot and root tissues of bothspecies, pea plants showed more severe toxic symptoms. The freshweight and percentage of water content of root and shoot decreasedconcomitantly to Cd supply. High Cd levels were found in thecell-wall fraction (Fraction I) and in Fraction IV (soluble)of maize plants, whereas Cd-treated pea accumulated more Cdin the soluble fraction. The protein concentration of FractionIV of pea shoot and root significantly increased upon treatmentwith 0.05 mM Cd, whereas maize showed no effect. Furthermore,a previously not visible protein ({small tilde}12 kDa), appearedin Fraction IV of pea root grown with the highest Cd supply.Cadmium treatment, in general, notably enhanced the concentrationsof 2-thiobarbituric acid reactive material (lipid peroxidationproducts) in pea fractions, presumably due to Cd-induced oxidativestress. Key words: Cadmium sensitivity, tissue fractions, stress, Pisum sativum, Zea mays