Further Transport of Metal Ions Inside Plants

For any of these organisms, delivery of ligands to soil or groundwater which then sequester metal ions (both essential and non-essential) requires decoupling of matter from some metabolic cycle. In simple cases, like with green plants, there is no demand for additional reactants to produce the corresponding ligands, as citrate or malate are taken from the tricarboxylate cycle directly. More generally speaking, however, formation of such extracellularly active ligands takes additional reactants which modify some precursor to obtain the moieties which in turn can coordinate some metal ion (e.g. phenolate, hydroxamate, amino groups), with the complex thus formed eventually being reabsorbed by the organism which emitted it for purposes of its own metal supplies. Fairly often, the additional reactant is an oxidant, e.g. dioxygen is needed to convert aromatics or monophenols into oli-gophenols, while NO or nitrate are required for making hydroxamates from 2-ketoenolates (hydroxylamine NH2OH which cleaves esters by direct nucleophilic substitution to yield hydroxamates, is not normally an intermediate of either biological NH3 oxidation or NOx-, N2 reductions. Apart from this, the hydroxam-ates which are produced by fungi or soil bacteria bear an alkyl or aryl group at N, accordingly would have to originate from N-functionalized hydroxylamines which are rare in biochemistry, also).

Citrate (or malate) is shuttled back into the tricar-boxylate cycle once the corresponding complexes have been resorbed by the plant roots; accordingly the complexes dissociate in a kind of oxidative ligand exchange, giving way to formation of new complexes of the absorbed metal ions combining with chelators in the cytosol. The equilibrium is shifted to the left by removal of the original ligand which had effected resorption. By evaporation in leaves a vertical water current is produced which also transports metal complexes through the shoot (stem) axis upward; thereafter the metal ions will get bound to nitrogeneous ligands like peptides and proteins. Metal transporters like the protein IRT-1 (IRT = iron regulated transporter) occur in epidermis cells next to the rhizosphere of plants and in the root/shoot interfacial region, e.g. in wheat or root epidermis cells of Arabidopsis thaliana (Clemens et al. 2002). Like in albumins of animals, IRT-1 effects metal binding by four histidine residues (i.e., imidazol ligands). In the former albumins metal ions such as Ni2+ are known to be rapidly removed from the protein sites (a matter of minutes at most) by simple addition of amino acids (Tabata and Sarkar 1992) which means k < 106 for nickel ions; accord-

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ingly the transporter is a rather poor ligand.

Homeostasis Plants

Fig. 2.6 Molecular mechanisms hold to explain accumulation of transition metal ions by and in plants. Letters (a) to (e) are to be taken in the same vertical arrangement in both plant and this picture, e.g. a = mobilization around the root, c = transport within the xylem. (a) metal ions get mobilized by secretion of chelators which in addition acidify the rhizosphere. (b) uptake of hydrated metal ions or (rather) their chelate complexes is augmented by various systems bound to the plasma membrane. (c) transport of transition metals from roots to shoot occurs via the xylem. Presumably the larger share is transported by means of the root symplast; an apoplastic passage in the root tips is also conceivable. After exchange (oxidative destruction) of the original ligands metals which made it into the xylem are other kinds of chelator complexes or else aquated ions. (d) After getting into the leaf apoplast several metals are bound to the

Fig. 2.6 Molecular mechanisms hold to explain accumulation of transition metal ions by and in plants. Letters (a) to (e) are to be taken in the same vertical arrangement in both plant and this picture, e.g. a = mobilization around the root, c = transport within the xylem. (a) metal ions get mobilized by secretion of chelators which in addition acidify the rhizosphere. (b) uptake of hydrated metal ions or (rather) their chelate complexes is augmented by various systems bound to the plasma membrane. (c) transport of transition metals from roots to shoot occurs via the xylem. Presumably the larger share is transported by means of the root symplast; an apoplastic passage in the root tips is also conceivable. After exchange (oxidative destruction) of the original ligands metals which made it into the xylem are other kinds of chelator complexes or else aquated ions. (d) After getting into the leaf apoplast several metals are bound to the various kinds of leaf cells and move among cells by means of the plasmodesmata. (e) Uptake into leaf cells is catalyzed by various carriers (not displayed). Trafficking of essential elements inside cells is effected by specialized, selective transporter proteins (metallochaperons) and carriers attached to endomembranes (nota bene: these latter processes occur in all cells rather than in leaves only). Symbols and abbreviations: CW = cell wall; M = metal; filled circles = chelators, filled oval dots = transporters, bean-shaped symbols = metallochaperons. Cp. Fig. 2.16 for transformation into a description focussed on subsequent biochemical changes of speciation, cp. Fig. 2.9 for the different manners in which fungi, mosses and vascular green plants use chemical elements and what they do to sequester them. Reproduced with permission by the author (from Clemens et al. 2002)

For selectivity of binding of metal ions, binding to carriers and its selectivity are relevant. Some ligands, production rates of which may be increased upon metal exposition, like nicotianamine (Fe, Cu), histidine (Ni), or citrate (Cd, Al) increase carryover of the said metal ions into the xylem in both hyperaccumulators and "normal" plants as do chelators which are not altered by metabolism, e.g. EDTA, when they are added to the soil. There is a difference in structure: whereas in albumines four histidines (or two histidines and two car-boxamides) are located at one end of the peptide sequence, they form a bridge between two helices in IRT-1 (Grossoehme et al. 2005). The limited complex formation stabilities of these ligand site arrays, allowing for reversible binding rather than the role of some "cation trap", is just suited for transportations tasks. Amino acids may "extract" the metal ions from this carrier (also others than Ni) by forming more stable complexes as is done in isolation of demetalated apo-proteins by EDTA addition (Noertemann 1999).

At low ligand concentrations, there will be an equilibrium among chelate complexes and aqua- or hydroxoions in the xylem. Usually, pH ยป 6 in the xylem; thus many aquaions get deprotonated including some forming (but weak) complexes (e.g., VO2+). Owing to free phosphate (H2PO4-) levels of up to several mmol/L, mobility of metals in the phloem likewise may differ markedly, being lowest of course for those metal ions which form hardly soluble phosphates (Li, Ca, Sr, Ba, Pb, Ag, etc.) and are immobile in the phloem, mobility being but slightly better for Mo, Fe, Mn, Co, Cu or Zn; conversely heavier alkali metals Na - Cs or Mg are pretty mobile there (Marschner 1986). Also sites where metal ions are located can differ in higher plants: some occur outside the cells mainly, becoming active parts of enzymes out there also (Ca, Cu, Al, [Mo]) while others reside and catalyze some processes either inside the cell (Mg, Fe, Co, Zn, Ni or Mn) or in cytoplasm (Mg, Co, Zn (Williams 1986)), being accompanied by several non-metals other than the liga-tion partners in each case (Si, Cl or B).

By relocating them via the phloem, plants can recover highly mobile ions such as K or Mg from e.g. aged leaves before these are dropped into the litter. This mechanism hampers a direct comparison of metal concentrations and distributions among different plant species (as was done before by correlation analysis (Markert 1996)) as transport or binding properties are likely to differ also. Apart from this, some elements less similar to each other than the REEs are also distinguished by very highly positive correlation coef ficients of their abundances in plants. Accordingly, pathways of transport and accumulation are likely to be very similar among these organisms as well. This prompted an attempt to quantify differences among the various plants, too (cp. Sections 3.2 and 4.2).

As a rule, green plants employ chemically less "sophisticated" species to take up metal ions via the roots than animals, fungi (e.g., yeasts) or bacteria do. In addition, plants change their environments and spe-ciation forms of metal ions which occur there less severely than both animals and chemolithoautotrophic bacteria do by both acidic leaching (stomach, Thiobacillus) and production/delivery/use of highly effective sequestering agents. Either process does not only change the environmental speciation thoroughly, with various soil organisms even being able to cause irreversible changes to the metal ion retention capacity of soils by complexation to polymers or in solid mineral phases (fungi, earth worms). When compared to this, metal distribution, partition and accumulation in plants should be more straightforward to understand, with the corresponding models being less complex than those applied for bioaccumulation in animals (cp. Paquin et al. 2003). Often alkaloids which are active in metal ion enrichment also pile up in photosynthetic organs for some period of time, e.g. the xanthine compounds caffeine, theobromine (Habermehl et al. 2003; Colacio-Rodriguez et al. 1983) and associated polyphenols such as caffeic acid in young leaves (leaf-tips) of tea (Camelia sinensis) or mate (Ilex paraguayensis). In older leaves concentrations get smaller, obviously in favour of releasing metals which were transported to the ultimate parts of a plant again while these parts get into using the metals. Of course, these and similar alkaloids, polyphenols are useful for accumulation of metal ions during plant development also, hence there may be stockpiles in seeds, e.g. caffeine (and caffeic acid) in coffee beans. Concerning cyanide, there is both production of cyanide (usually from glycine precursor) and conversion into other ligands (asparagine via 3-cyanoalanine) in small roots (Ting and Zschoche 1970), contributing to chemistry of metal uptake. As the nitrile 3-cyanoalanine and carboxamide asparagine are formed from cysteinate, there also is a considerable increase in EL(L) of the third (terminal) donor site of the amino acid, changing selectivities in a similar way like cyanide does.

A look aside to other groups of elements and organisms: the realm of chemical ecology and food chain shaping.

Substantial enrichments of radionuclides are known to occur with aquatic plants (Nuclear Task Force 1996; Weltje 2003), and REE ions which usually have highly negative x1d and also x2d values (even being rather identical for Y(III), Ce(III)) get thoroughly enriched there. For distributions of cerium (141Ce autoradiography) in a terrestrial plant see Guo et al. 2007. This tendency for pronounced metal enrichment of course extends to organisms which even use metals as electron sinks or sources of metabolism, like Fe-oxidizing bacteria Leptothrix ochra-cea and implies binding partners to have EL(L) << 0 V (cp. Figs. 2.1 and 2.9). For L.ochracea, EL(L)eff = -0.16 V for a total analysis including biogenic oxide concretions.

There are several methods for directly depicting metal distributions in plants, including autoradiogra-phy and X-ray fluorescence (XRF or XFS). The former method - essentially the procedure by which Becquerel discovered the phenomenon of radioactivity in 1896 -means using and detecting (by photographical means) emission by some radionuclide (often non-metals such as UC, 14C, 32P but also 55;59Fe and 109Cd (Figs. 2.7 and 2.8) in some instances) which was administered to the plant before. This will produce a quantitative picture of the spatial distribution of radiation emitted within the organism or in another kind of sample (by darkening of some conventional AgHal-based photographic film) whereas XRF can be done on the untreated plant making use of synchrotron radiation for excitation of stable nuclei also. XRF can also afford - by EXAFS (shifts of typical fluorescence energies) - some information on the ligand sphere around the metal ions; in addition to avoiding radionuclide uses and any other prior sample treatment (but not omitting radiation), it is a multielement method. How different metal distributions can be although there are similar maxima in the upper root region and decreases along the shoot, Figs. 2.7 and 2.8 (the latter by Feller 2005) do show:

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