Organic acids (OAs) have been associated with metal hyperaccumulation and tolerance in a range of plant species and have been proposed as important cellular ligands for Zn, Cd and Ni (Salt et al. 1999; Kupper et al. 2004). The carboxylic acids known to be present in high concentrations in the cell vacuoles of photosyn-thetic tissues include citric, isocitric, oxalic, tartaric, malic, malonic and aconitic (Callahan et al. 2007). Many studies have implied that these acids play a role in hyperaccumulation (Rauser 1999; Salt et al. 1999; Romheld and Awad 2000; Chiang et al. 2006). Analysis of tissues from metal hyperaccumulator species using X-ray absorption techniques has identified OAs as the predominant ligands. By X-ray absorption spectrometry (XAS) and extended X-ray absorption fine structure (EXAFS) analysis, citrate was identified as the predominant ligand for Zn in leaves of Thlaspi caerulescens (Salt et al. 1999). Similarly, Ni-citrate accounted for one-quarter of the Ni species in leaves of the Ni hyperaccumulator T. goesingense and in the related nonaccumulator T. arvense (Kramer et al. 2000). The identification of the vacuole as the major subcellular compartment for Zn, Cd and Ni and the favoring of the formation of metal-OA complexes in the acidic environment of the vacuolar lumen suggest that citrate and malate are probably relevant only as ligands for these metals within vacuoles (Kramer et al. 2000; Ma et al. 2005).
Studies have demonstrated that the primary constituents of root exudates are low-molecular weight organic acids (LMWOAs) that play essential roles in making sparingly soluble soil Fe, P, and other metals available to growing plants (Romheld and Awad 2000). Acetic, lactic, glycolic, malic, maleic, and succinic acids were found in rhizosphere soils of tobacco and sunflower (Chiang et al. 2006). Concentrations of these LMWOAs exudates increased with increasing amendment of Cd concentrations in the rhizosphere soils. After the loss of H+, each acid contains a COO- group, which binds to the cations. Correlation coefficients between concentrations of Cd amendment versus LMWOAs exudates of tobacco and sunflower were 0.85 and 0.98, respectively (Chiang et al. 2006). Positive correlations have been found between external Zn and organic acid concentrations in the roots of hyperaccumulator plants A. halleri (Zhao et al. 2000). These results suggest that the different levels of LMWOAs present in the rhizosphere soil may play an important role in the solubilization of heavy metals that bind with soil particles into soil solution and followed by uptake by plants. However, this mechanism does not draw a sharp line between toxic and essential metals for uptake and further utilization. This role may be covered by other specific biological ligands or transporters in the root and shoot tissues.
Nicotianamine (NA), a non-proteinaceous amino acid synthesized in all plants by the condensation of three S-adenosyl-methionine molecules through the activity of the enzyme nicotianamine synthase (NAS), is ubiquitously present in higher plants (Fig. 1). It is known to be involved in chelation of metals such as Fe, Cu, Zn for their enhanced extraction by roots and/or transport to shoost, especially under mineral-deficient conditions (Takahasi et al. 2003; Mari et al. 2006). However, recent evidence supports their possible functions in heavy metal-tolerance and hyperaccumulation in plants. The hyperaccumulation of Zn and Cd is a constitutive property of the metallophyte A. halerii. Recently, Weber et al. (2004) have used Arabidopsis gene chips to identify those genes that are more active in roots of A. halleri than A. thaliana under controlled conditions. Two genes showing highest levels of expression in A. halleri roots code for a NAS and a putative Zn2+ uptake system. In addition, roots of A. halleri also show higher levels of both NA and NAS. A. halleri presents a 2-fold increase of its NA root content probably linked to the constitutive expression of the AhNAS2 gene. Expression of NAS in S. pombe cells has demonstrated that formation of NA can confer Zn2+ tolerance. Taken together, these observations suggest active roles of NA in plant Zn homeostasis and NAS in hyperaccumulation of Zn in A. halleri (Weber et al. 2004). Recently, it was reported that the overexpression of TcNAS in A. thaliana transgenic plants also confers Ni resistance (Pianelli et al. 2005), strengthening the idea that NA could play a role in metal tolerance and hyperaccumulation.
Plant cells contain many other small organic ligands with variable functional groups, including amino acids, polyamines, nucleotides, phytates and other phosphate sugars. Of these, polyamines appear to act as a messenger or a molecule to stabilize or protect the cell membranes rather than as direct binding ligands to toxic heavy metals (Sharma and Dietz 2006). Nucleotides, phytates and sugar phosphates can conjugate to Ca, Mn, Mg, Al and other metals through their O-bonds. Especially, the importance of phytates in coordination and storage of phosphate and metals such as Zn, Mg, and K in vacuole and cytoplasm and also in the detoxification of Cd has been widely suggested (Van Steveninck et al. 1992; Hayden and Cobbett 2006). Amino acids are the most abundant amphoteric ions with variable forms and residues, existing in 10-100 mM orders of concentrations and serving multiple functions in plant cells. Cysteine (Cys) is a thiol compound that has a S-donor residue equivalent to a GSH molecule. However, its internal level does not usually exceed that of GSH or PCs, probably because of the restricted supply of total S available for it and its quick turnover and utilization for the other thiol ligands and proteins. Acidic amino acids, glutamic acid (Glu) and aspartic acid (Asp), provide an extra carboxyl group (—COOH), and their amides, glutamine (Gln) and asparagine (Asn), provide an acid amide group consisting of both O- and N-donors (—CO-NH2). All these are generally rich in phloem sap, for example, at near 300 mM in cereals and 50 mM in some dicotyledonous plants (Oshima et al. 1990; Winter et al. 1992), and can be potential ligands for translocational metal cations. Histidine (His) is the most characterized imidazole (=NH)-containing amino acid that plays a central role in binding to and transport of Ni, especially in Ni-hyper-accumulating plants (Kramer et al. 2000; Callahan et al. 2006). Two His molecules can make a stable complex chelating to one Ni (Callahan et al. 2006). Furthermore, proline (Pro) has been most extensively studied for its unique and important function as a compatible solute in many plants affected by water-deficit and salinity stress, but interestingly, heavy metals such as Cu, Cd, Zn or Pb also significantly stimulate the accumulation and/or biosynthesis of Pro in many plants (Sharma and Dietz 2006). Possible roles of Pro as a direct N-donor ligand conjugating to heavy metals are not established as yet, but will be more attractive in combination with its role as osmotic protectant or antioxidant under complex conditions including salinity and drought stress.
As mentioned above, there are possible interactions between different soluble organic ligands and different metals in cytoplasm, vacuole and other apoplastic solutions in shoots and roots. These solutions also contain inorganic anions such as sulfate, phosphate, nitrate, borate, carbonate, chloride and silicate. These inorganic anions and counterpart cations affect the organic ligand's interactions with metals in each site at different but almost constant pH conditions (Callahan et al. 2006). Some bindings between metals and ligands are not specific and not stable, especially under varied pH and ion-strength conditions. Conversely the regulated conditions can promise a unique and established mechanism for metal transport and binding systems in land plants.
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