Dieter Neumann

Tolerance to toxic metals by a gene family of phytochelatin synthases from plants and yeast

Phytochelatins play major roles in metal detoxification in plants and fungi. However, genes encoding phytochelatin synthases have not yet been identified. By screening for plant genes mediating metal tolerance we identified a wheat cDNA, TaPCS1, whose expression in Saccharomyces cerevisiae results in a dramatic increase in cadmium tolerance. TaPCS1 encodes a protein of ∼55 kDa with no similarity to proteins of known function. We identified homologs of this new gene family from Arabidopsis thaliana, Schizosaccharomyces pombe, and interestingly also Caenorhabditis elegans. The Arabidopsis and S.pombe genes were also demonstrated to confer substantial increases in metal tolerance in yeast. PCS‐expressing cells accumulate more Cd2+ than controls. PCS expression mediates Cd2+ tolerance even in yeast mutants that are either deficient in vacuolar acidification or impaired in vacuolar biogenesis. PCS‐induced metal resistance is lost upon exposure to an inhibitor of glutathione biosynthesis, a process necessary for phytochelatin formation. Schizosaccharomyces pombe cells disrupted in the PCS gene exhibit hypersensitivity to Cd2+ and Cu2+ and are unable to synthesize phytochelatins upon Cd2+ exposure as determined by HPLC analysis. Saccharomyces cerevisiae cells expressing PCS produce phytochelatins. Moreover, the recombinant purified S.pombe PCS protein displays phytochelatin synthase activity. These data demonstrate that PCS genes encode phytochelatin synthases and mediate metal detoxification in eukaryotes.

Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron

Iron (Fe) is necessary for all living cells, but its bioavailability is often limited. Fe deficiency limits agriculture in many areas and affects over a billion human beings worldwide. In mammals, NRAMP2/DMT1/DCT1 was identified as a major pathway for Fe acquisition and recycling. In plants, AtNRAMP3 and AtNRAMP4 are induced under Fe deficiency. The similitude of AtNRAMP3 and AtNRAMP4 expression patterns and their common targeting to the vacuole, together with the lack of obvious phenotype in nramp3‐1 and nramp4‐1 single knockout mutants, suggested a functional redundancy. Indeed, the germination of nramp3 nramp4 double mutants is arrested under low Fe nutrition and fully rescued by high Fe supply. Mutant seeds have wild type Fe content, but fail to retrieve Fe from the vacuolar globoids. Our work thus identifies for the first time the vacuole as an essential compartment for Fe storage in seeds. Our data indicate that mobilization of vacuolar Fe stores by AtNRAMP3 and AtNRAMP4 is crucial to support Arabidopsis early development until efficient systems for Fe acquisition from the soil take over.

Cytoplasmic heat shock granules are formed from precursor particles and are associated with a specific set of mRNAs.

In heat-shocked tomato cell cultures, cytoplasmic heat shock granules (HSGs) are tightly associated with a specific subset of mRNAs coding mainly for the untranslated control proteins. This messenger ribonucleoprotein complex was banded in a CsCl gradient after fixation with formaldehyde (approximately 1.30 g/cm3). It contains all the heat shock proteins and most of the RNA applied to the gradient. During heat shock, a reversible aggregation of HSGs from 15S precursor particles can be shown. These pre-HSGs are not identical to the 19S plant prosomes. Ultrastructural analysis supports the ribonucleoprotein nature of HSGs and their composition of approximately 10-nm precursor particles. A model summarizes our results. It gives a reasonable explanation for the striking conservation of untranslated mRNAs during heat shock and may apply also to animal cells.

Silicon and heavy metal tolerance of higher plants

The heavy metal tolerant Cardaminopsis halleri, grown on Zn and Cu polluted soil, showed electron dense metal containing precipitates (Zn, Cu, Sn, Fe, Al) on the leaf surface, in the intercellular spaces (Zn, Cu, Sn), the cell walls and the cell wall thickenings of the xylem vessels (Zn, traces of Cu and Fe). Large amounts of Zn were measured in the vacuoles, the main storage compartment for this metal in Cardarminopsis. The cytoplasm and nuclei contained small precipitates, including mainly Zn and Si. As shown by ESI Zn was co-localized with Si in these structures. The EEL-spectra of the cytoplasmic precipitates corresponded with the spectra of Zn-silicate. Besides Zn-silicate, electron translucent structures in the cytoplasm were identified as SiO2 by their EEL spectra. It was concluded that in the cytoplasm of Cardaminopsis Zn is transiently accumulated as silicate, being slowly degraded to SiO2. Zn is translocated into the vacuole and accumulated in an unknown form. A second Si and Zn-uptake mechanism was found, excluding a membrane and cytoplasm passage. Pinocytotic vesicles, formed by the plasmamembrane and the tonoplast, enable a direct translocation of Si and Zn from extracellular compartments into the vacuole. The formation of Zn-silicate is part of the heavy metal tolerance mechanism and may be responsible for the amelioration of the Zn toxicity in Cardaminopsis.

Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves.

Biochemical and electron microscopic analyses of heat-shocked suspension cultures of Peruvian tomato (Lycopersicon peruvianum) revealed that a considerable part of the dominant small heat shock proteins (hsps) with an Mr of approximately 17,000 are structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depends on heat shock conditions (37 to 40 degrees C) and the presence or simultaneous synthesis of hsps. However, under certain conditions, e.g., in preinduced cultures maintained at 25 degrees C, hsps also accumulate as soluble proteins without concomitant assembly of heat shock granules. Similar heat shock-induced cytoplasmic aggregates were also observed in other cell cultures and heat-shocked tomato leaves and corn coleoptiles.

Heat-shock proteins induce heavy-metal tolerance in higher plants

Cell cultures of Lycopersicon peruvianum L. stressed with CdSO4 (10−3M) show typical changes in the ultrastructure, starting with the plasmalemma and later on extending to the endoplasmic reticulum and the mitochondrial envelope. Part of the membrane material is extruded, with the formation of osmiophilic droplets which increase in size and number during the stress period. After 4 h, about 20‰ of the cells are dead. A short heat stress preceeding the heavy-metal stress induces a tolerance effect by preventing the membrane damage. The cells show a normal ultrastructure with one exception: cytoplasmic heat-shock granules are formed. This protective effect can be abolished by cycloheximide. Cadmium uptake is not markedly influenced by the heat stress. Cadmium is found together with sulfur in small deposits in the vacuoles of stressed cells. The precipitates contain an excess of sulfur, evidently due to the stress-induced formation of phytochelatins. The role in heavy-metal tolerance of heat-shock proteins in the plasmalemma (HSP70) and in cytoplasmic heat-stress granules (HSP17, HSP70) is discussed.

How does Armeria maritima tolerate high heavy metal concentrations

Summary The perennial plant Armeria maritima ssp. halleri , growing on the copper-rich soil of a medieval mine dump, tolerates high heavy metal (HM) concentrations and accumulates e.g. 2000 (leaves) to 4000 (roots) times more copper in comparison to plants growing under normal conditions. For copper, as an example, the mechanisms of the HM-tolerance are discussed. The HM-tolerance of Armeria is the result of morphological differentiations and biochemical alterations. As measured by EDX analysis a great part of the copper in roots and leaves is retained in vacuoles of idioblasts («tannin cells») containing homogeneous or flaky precipitates of osmiophilic material. In the homogeneous precipitates, high copper concentrations can be measured. EELS spectra revealed that the copper in these vacuoles is chelated by polyhydroxy phenolic compounds. Moreover, a complex mixture of phenolic compounds from leaves and roots of Armeria can be separated by HPLC. Copper ions reaching the vascular bundle are translocated via the transpiration stream into the leaves and are excreted partly by salt glands on both leaf surfaces. Crystals on the surface of the leaves contain besides P, S, Cl, K and Ca large amounts of Cu and to a smaller extent Zn, Ni, Fe and Mn. A significant part of the copper in roots and leaves is found to be localized in cell walls, in the cytoplasm, in the stroma of the plastids and in the chromatin of the nuclei. In these compartments the copper is bound preferentially to proteins, as determined by EELS. Obviously, the copper in the cytoplasm represents a stress situation for the cell, resulting in an expression of heat stress proteins (HSP).

Phytochelatins and heavy metal tolerance

The induction and heavy metal binding properties of phytochelatins in heavy metal tolerant (Silene vulgaris) and sensitive (tomato) cell cultures, in water cultures of these plants and in Silene vulgaris grown on a medieval copper mining dump were investigated. Application of heavy metals to cell suspension cultures and whole plants of Silene vulgaris and tomato induces the formation of heavy metal‐phytochelatin-complexes with Cu and Cd and the binding of Zn and Pb to lower molecular weight substances. The binding of heavy metal ions to phytochelatins seems to play only a transient role in the heavy metal detoxification, because the Cd- and Cu-complexes disappear in the roots of water cultures of Silene vulgaris between 7 and 14 days after heavy metal exposition. Free heavy metal ions were not detectable in the extracts of all investigated plants and cell cultures. Silene vulgaris plants grown under natural conditions on a mining dump synthesize low molecular weight heavy metal binding compounds only and show no complexation of heavy metal ions to phytochelatins. The induction of phytochelatins is a general answer of higher plants to heavy metal exposition, but only some of the heavy metal ions are able to form stable complexes with phytochelatins. The investigation of tolerant plants from the copper mining dump shows that phytochelatins are not responsible for the development of the heavy metal tolerant phenotypes. # 1999 Published by Elsevier Science Ltd. All rights reserved.

Heavy metal tolerance of Silene vulgaris

Summary Silene vulgaris ssp. humilis , a heavy metal tolerant plant growing on the polluted soil of a medieval copper mining dump, accumulates considerable amounts of heavy metals (HM) in its roots and shoots. The intracellular distribution of HMs in the leaves was investigated by conventional and analytical (EDX, ESI, EELS) electron microscopy. Part of the HMs, Fe, Cu, and Zn occur as crystalline compounds on the surface of the leaves. The epidermal cell walls accumulate Fe, Ni, Cu, AI, Sn, and Zn. Cu within the cell walls is tightly bound to a protein with oxalate oxidase activity, evidencing a high homology to germin. Zn and Sn are accumulated in the cell walls as silicate. Cytoplasm and organelles contain only traces of Cu and Sn, while in the vacuoles no HMs are detected. In the epidermal cell walls, intercellular spaces, and in vacuoles there are high concentrations of Si, forming crystal-like structures. EELS and quantum-chemical calculations reveal these structures as SiO 2 . The role of Si in the HM-tolerance of Silene is discussed.

Heavy metal tolerance of Minuartia verna

Summary Individuals of Minuartia verna ssp. hercynica, growing on heavy metal polluted soil of medieval mine dumps, accumulate remarkable concentrations of copper and zinc in their leaves. The cellular and intracellular distributions of heavy metals were investigated by conventional electron microscopy, EDX, ESI and EELS. Considerable amounts of Fe, Cu, Zn, and Al found on the leaf surface are excreted by hydrathodes. Intracellular spaces and cell walls of the leaf parenchyma contain Fe, Cu, Zn, and Pb, whereas no metals could be detected in the cytoplasm, vacuole or cellular organelles. High Si concentrations evident in vacuoles, cell walls and intercellular spaces are not directly involved in the detoxification of the heavy metals with one exception, zinc is precipitated as a Zn-silicate in the epidermal cell walls. The possible mechanisms of heavy metal tolerance in Minuartia and the role of Si in these processes are discussed.