The array of Fe-regulated transporters suggests a complex network of intra- and inter-cellular Fe trafficking, leading to the homeostasis of Fe according to the needs of the plant (Schmidt, 2003). As mentioned above, little is known about the molecular mechanisms of the interactions between heavy metals and Fe during their influx to the cytoplasm, but even less data concerning heavy metal interaction with Fe regulation inside the cell are available. It was found that under Fe-starvation the disruption of AtNRAMP3 led to increased accumulation of Mn and Zn in the roots, whereas its over expression down-regulates Mn accumulation and also the expression of IRT1 and FRO2 responsible for high affinity Fe uptake (Thomine et al., 2003). The authors suggested that AtNRAMP3 localized in the vacuolar membrane influences metal accumulation and Fe uptake by mobilizing vacuolar metal pools (Fe, Cd and other metals) to the cytosol.
The intracellular level of Fe and other heavy metals are by all means strictly regulated, and different substances are involved in the process. Nicotianamine (NA), a non-proteogenic amino acid ubiquitous in higher plants, seems to be the principal chelator of Fe within the cell when Fe is not bound to target molecules such as heme or stored as phytoferritin (Hell and Stephan, 2003). Nicotianamine may function in keeping Fe soluble and available as well as preventing Fenton reactions, which lead to oxygen radical formation when free Fe2+ ions are present (von Wiren et al., 1999). Organic acids and amino acids are also available chelating agents for Fe, although the stability constants of those complexes are high enough only for Fe3+ (see Table 6-1 for citrate).
Phytochelatins (PCs) are Cys-rich molecules with the structure (y-Glu-Cys)n-Gly, where n is the number of repetition of the unit (usually 2-11). Phytochelatins chelate heavy metals, such as Cu, Cd and Hg, which have high affinity with the thiolic groups of Cys, and play a crucial role for their sequestration into the vacuole. These compounds are synthesized from glutathione by the enzyme PC synthase. This mechanism may be the most significant response of plant cells to heavy metal toxicity, because the PC synthase is induced promptly and greatly reduces free heavy metal ion concentrations in the cytoplasm (Rauser, 1995).
Under heavy metal toxicity, the synthesis of another group of Cys-rich peptides has been reported. These peptides are analogous to the gene-encoded metallothioneins found in animals, fungi and cyanobacteria, which have a role in complexing and detoxifying different kinds of heavy metals. In plants, metallothionein synthesis has been reported to be induced by several heavy metals, including Cu in Arabidopsis thaliana (Murphy et al., 1997), Zn in wheat (Lane et al., 1987) and Cd in soybean (Blakely et al., 1986).
The synthesis of potential chelating agents in the cytoplasm may raise the question of competition between metals for organic ligands, especially under conditions of hyperaccumulation. Although the basic factors determining the formation of complexes are thermodynamic stability and concentrations, the prediction of the true complexes occurring at cytoplasmic pH should also consider other aspects. Von Wirén et al. (1999) investigated experimentally the formation of NA complexes with Fe2+ and Fe3+, and found that although the formation of Fe(III)-NA is thermodynamically favoured, under aerobic conditions the Fe(II)-NA complex is more stable kinetically. Kinetic considerations are also of outmost importance in the formation of other chelate complexes (e.g. with DMA) with different metals. Due to slow kinetics, the formation of Cr(III)-DMA complex is restricted. This demonstrates that metal complexes may preferentially form if favoured by fast kinetics, even if their formation constants are less favorable (Hider 1984).
Concerning the effect of heavy metals on Fe homeostasis, the observations presented above imply that complex formation may be shifted from one ligand to another. For example, it is known that there are special proteins binding and escorting Cu to the appropriate targets. These copper chaperones may function under conditions of normal Cu supply (Field et al., 2002). However at elevated concentrations, as more and more Cu ions are available in the cytoplasm, they may form complexes with ligands such as amino acids and NA (Herbik et al., 1996). As PC synthesis is switched on, Cu would also bind to PCs. In yeast exposed to Ni, Ni(II)-NA complexes have been determined (Vacchina et al., 2003). In Fe deficient sugar beet plants, the level of glutathione, a precursor of PCs, increased 1.6-fold (Zaharieva and Abadía, 2003), whereas under Cd toxicity, which is thought to induce Fe deficiency, PC synthesis is increased (Sanitá di Toppi and Gabrielli, 1999). The transient competition between heavy metals and Fe for chelating agents may substantially modify Fe homeostasis. Complex formation, even if kinetically favoured, is influenced by equilibrium processes, in which thermodynamic stability is the prevalent factor. Also, physiological uptake, transport, assimilation and sequestration processes continuously alter metal and ligand concentrations in the cytosol. For these reasons, direct analysis of metal-chelates existing in plant materials should be strongly encouraged, instead of relying only on theoretical chemical speciation (Hider et al., 2004).
The appearance of non-essential heavy metals in the shoot gave rise to the hypothesis of apoplastic transport (Bell et al., 1991a; White 2001). This pathway has been suggested as a possible rout for Zn hyperaccumulation in Thlaspi caerulescens (White et al., 2002). However, in more recent studies, circumstancial and experimental evidence have been presented against a substantial role for apoplastic metal loading (Ernst et al., 2002; Kerkeb and Krämer, 2003). Thus, the translocation of heavy metals as well as Fe likely starts with xylem loading from the symplasm. The molecular background of xylem loading of heavy metals and Fe is still poorly known. There is, however, substantial analytical data on the concentrations and forms of metals present in the xylem sap. It is well established that Fe is translocated as Fe3+ complexed by citrate (Tiffin, 1966). Citrate was also found to be the principal compound chelating heavy metals in the xylem sap of different plant species exposed to Pb or Cd (Senden et al., 1995; Tatar et al., 1998). Concerning the transport of Fe into the xylem, a shift between chelating ligands may occur, since at pH 5.5-6 Fe can be readily released by NA to favour the formation of citrate complexes such as those predicted by chemical speciation studies [FeCitrateOH]-1 and [FeCitrate2]-3 (Lopez-Millan et al., 2000). However, since the prevalent Fe form in the cytoplasm could be Fe(II)-NA as indicated previously, xylem loading should theoretically involve an oxidation step to decrease the stability of the complex. Nevertheless, NA was also shown to be present in the xylem sap and was suggested to have a role in Cu transport (Pich et al., 1994; Liao et al., 2000). Obviously, heavy metals may also interact with Fe during transport, modifying their uptake, accumulation and physiological effect in the leaf, especially when they are present at elevated concentrations, such as in cases of hyperaccumulation.
As chelate-assisted phytoextraction requires the application of chelating agents at high concentrations, the transported metal form may change as more and more chelating agents enter the plant. Since membrane integrity may be diminished, at least partially, apoplastic xylem loading may be significant in these cases. Direct measurement of Pb-EDTA in xylem exudates of Brassica juncea confirmed that the majority of Pb was transported as Pb-EDTA (Vassil et al., 1998; Epstein et al., 1999).
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