Toxic effects of both essential and non‐essential heavy metals are well documented in plants. Very little is known, however, about their modes of toxicity, about tolerance mechanisms and the signalling cascades involved in mediating transcriptional responses to toxic metal excess. We analysed transcriptome changes upon Cd2+ and Cu2+ exposure in roots of Arabidopsis thaliana and the Cd2+‐hypertolerant metallophyte Arabidopsis halleri . Particularly, three categories of genes were identified with the help of this comparative approach: (1) common responses, which might indicate stable and functionally relevant changes conserved across plant species; (2) metallophyte‐specific responses as well as transcripts differentially regulated between the two species, representing candidate genes for Cd2+ hypertolerance; and (3) those specifically responsive to Cd2+ and therefore indicative of toxicity mechanisms or potentially involved in signalling cascades. Our data define, for instance, Arabidopsis core responses to Cd2+ and Cu2+. In addition, they suggest that Cd2+ exposure very rapidly results in apparent Zn deficiency, and they show the existence of highly specific Cd2+ responses and distinct signalling cascades. Array results were independently confirmed by real‐time quantitative PCR, thereby further validating cross‐species transcriptome analysis with oligonucleotide microarrays.

Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+‐hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray‐ionization‐quadrupole‐time‐of‐flight mass spectrometry we prove the formation of nicotianamine in N. crassa . Transcript level is strongly upregulated under Zn deficiency as shown by real‐time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions.

Tobacco (Nicotiana tabacum L. cv Xanthi) plants were exposed to toxic levels of zinc (Zn). Zn exposure resulted in toxicity signs in plants, and these damages were partly reduced by a calcium (Ca) supplement. Confocal imaging of intracellular Zn using Zinquin showed that Zn was preferentially accumulated in trichomes. Exposure to Zn and Zn + Ca increased the trichome density and induced the production of Ca/Zn mineral grains on the head cells of trichomes. These grains were aggregates of submicrometer-sized crystals and poorly crystalline material and contained Ca as major element, along with subordinate amounts of Zn, manganese, potassium, chlorine, phosphorus, silicon, and magnesium. Micro x-ray diffraction revealed that the large majority of the grains were composed essentially of metal-substituted calcite (CaCO3). CaCO3 polymorphs (aragonite and vaterite) and CaC2O4 (Ca oxalate) mono- and dihydrate also were identified, either as an admixture to calcite or in separate grains. Some grains did not diffract, although they contained Ca, suggesting the presence of amorphous form of Ca. The presence of Zn-substituted calcite was confirmed by Zn K-edge micro-extended x-ray absorption fine structure spectroscopy. Zn bound to organic compounds and Zn-containing silica and phosphate were also identified by this technique. The proportion of Zn-substituted calcite relative to the other species increased with Ca exposure. The production of Zn-containing biogenic calcite and other Zn compounds through the trichomes is a novel mechanism involved in Zn detoxification. This study illustrates the potential of laterally resolved x-ray synchrotron radiation techniques to study biomineralization and metal homeostasis processes in plants.

Cadmium is a major environmental pollutant that enters human food via accumulation in crop plants. Responses of plants to cadmium exposure—which directly influence accumulation rates—are not well understood. In general, little is known about stress-elicited changes in plants at the proteome level. Alterations in the root proteome of hydroponically grown Arabidopsis thaliana plants treated with 10 μM Cd2+ for 24 h are reported here. These conditions trigger the synthesis of phytochelatins (PCs), glutathione-derived metal-binding peptides, shown here as PC2 accumulation. Two-dimensional gel electrophoresis using different pH gradients in the first dimension detected on average ∼1100 spots per gel type. Forty-one spots indicated significant changes in protein abundance upon Cd2+ treatment. Seventeen proteins found in 25 spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Selected results were independently confirmed by western analysis and selective enrichment of a protein family (glutathione S-transferases) through affinity chromatography. Most of the identified proteins belong to four different classes: metabolic enzymes such as ATP sulphurylase, glycine hydroxymethyltransferase, and trehalose-6-phosphate phosphatase; glutathione S-transferases; latex allergen-like proteins; and unknown proteins. These results represent a basis for reverse genetics studies to better understand plant responses to toxic metal exposure and to the generation of internal sinks for reduced sulphur.

Monochlorobimane was used as a model xenobiotic for Arabidopsis to directly monitor the compartmentation of glutathione‐bimane conjugates in situ and to quantify degradation intermediates in vitro. Vacuolar sequestration of the conjugate was very fast and outcompeted carboxypeptidation to the γ‐glutamylcysteine‐bimane intermediate (γ‐EC‐B) by phytochelatin synthase (PCS) in the cytosol. Following vacuolar sequestration, degradation proceeded to cysteine‐bimane without intermediate. Only co‐infiltration of monochlorobimane with Cd2+ and Cu2+ increased γ‐EC‐B formation to 4% and 25%, respectively, within 60 min. The role of PCS under simultaneous heavy metal stress was confirmed by investigation of different pcs1 null‐mutants. In the absence of elevated heavy metal concentrations glutathione‐conjugates are therefore first sequestered to the vacuole and subsequently degraded with the initial breakdown step being rate‐limiting.

Over the past 200 years emissions of toxic heavy metals have risen tremendously and significantly exceed those from natural sources for practically all metals. Uptake and accumulation by crop plants represents the main entry pathway for potentially health-threatening toxic metals into human and animal food. Of major concern are the metalloids arsenic (As) and selenium (Se), and the metals cadmium (Cd), mercury (Hg), and lead (Pb). This review discusses the molecular mechanisms of toxic metal accumulation in plants and algae, the responses to metal exposure, as well as our understanding of metal tolerance and its evolution. The main emphasis will be on cadmium, which is by far the most widely studied of the non-essential toxic metals/metalloids. Entry via Zn2+, Fe2+, and Ca2+ transporters is the molecular basis of Cd2+ uptake into plant cells. Much less is known about the partitioning of non-essential metals and about the genes underlying the enormous diversity among plants with respect to Cd accumulation in different tissues. Numerous studies have described symptoms and responses of plants upon toxic metal exposure. Mysterious are primary targets of toxicity, the degree of specificity of responses, the sensing and the signaling events that lead to transcriptional activation. All plants apparently possess a basal tolerance of toxic non-essential metals. For Cd and As, this is largely dependent on the phytochelatin pathway. Not understood is the molecular biology of Cd hypertolerance in certain plant species such as the metallophytes Arabidopsis halleri or Thlaspi caerulescens.

Both essential and non-essential transition metal ions can easily be toxic to cells. The physiological range for essential metals between deficiency and toxicity is therefore extremely narrow and a tightly controlled metal homeostasis network to adjust to fluctuations in micronutrient availability is a necessity for all organisms. One protective strategy against metal excess is the expression of high-affinity binding sites to suppress uncontrolled binding of metal ions to physiologically important functional groups. The synthesis of phytochelatins, glutathione-derived metal binding peptides, represents the major detoxification mechanism for cadmium and arsenic in plants and an unknown range of other organisms. A few years ago genes encoding phytochelatin synthases (PCS) were cloned from plants, fungi and nematodes. Since then it has become apparent that PCS genes are far more widespread than ever anticipated. Searches in sequence databases indicate PCS expression in representatives of all eukaryotic kingdoms and the presence of PCS-like proteins in several prokaryotes. The almost ubiquitous presence in the plant kingdom and beyond as well as the constitutive expression of PCS genes and PCS activity in all major plant tissues are still mysterious. It is unclear, how the extremely rare need to cope with an excess of cadmium or arsenic ions could explain the evolution and distribution of PCS genes. Possible answers to this question are discussed. Also, the molecular characterization of phytochelatin synthases and our current knowledge about the enzymology of phytochelatin synthesis are reviewed.

Nutritional micronutrient deficiencies and exposure to pollutant metals threaten human health globally. Plant crops are at the beginning of a food chain that largely determines food metal contents. In order to survive, all organisms have to supply appropriate amounts of each micronutrient to the correct target apometalloproteins and at the same time avoid adventitious metal binding to non-target metal binding sites or other cellular compounds. This requires the operation of metal homeostasis networks, which orchestrate the mobilization, uptake, distribution, intracellular trafficking, chelation, and sequestration of all metal ions. Presumably as a result of time-dependent and local variations in bioavailable soil metal concentrations, plant metal homeostasis networks exhibit a remarkably high degree of plasticity and natural diversity. This is a review covering the current knowledge of metal-dependent processes and proteins, metal homeostasis and its regulation, and the molecular mechanisms underlying naturally selected metal hypertolerance and metal hyperaccumulation in higher plants.

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