Nonspecific Metal Chelating Agents Low Molecular Weight Organic Acids

Low molecular weight (LMW) organic acids, along with siderophores, are among the main mediators of the biological weathering of soils caused by living organisms, leading to the dissolution of a variety of minerals through a double effect of metal cation chelation and anion displacement by protonation (Landeweert et al. 2001; Gadd 2007; van Scholl et al. 2008). In soils, organic acids come mainly from plants that release them into the rhizosphere by exudation from their roots, as well as from fungi and bacteria, and chemically they are carboxylic acids, with one or more functional groups per molecule (e.g., formic, malic, oxalic, citric, succinic, etc.). Those acids that present more than one carboxylic group (di- or tricarboxylic) are important in the formation of complexes with metal ions and can be considered as metal-chelating agents. Under the normally existing pH conditions in biological systems, these acids are found in the form of carboxylate anions (e.g., oxalate, citrate, etc.).

Citric (tricarboxylic) and oxalic (dicarboxylic) acids have been the most investigated in plants and fungi, mainly in saprophytes and pathogens, due not only to their nutritional and physiological importance for these organisms, but also to their industrial importance. Oxalate can form complexes with a variety of trivalent (e.g., Fe3+, Al3+, Cr3+) and divalent (e.g., Ca2+, Cu2+, Mg2+, Zn2+, Pb2+) metal ions and also with actinides and lanthanides; some oxalates are soluble, like that of Fe3+, but others can be insoluble like those of Ca2+ and Pb2+. Similarly, the citrate can form different complexes with a variety of metal ions (e.g., Ca2+, Cd2+, Cu2+, Pb2+, Fe3+) (Dutton and Evans 1996; Jones 1998; Gadd 1999).

LMW organic acids perform important and varied metabolic roles for living cells and also participate in the mobilization and uptake of a series of mineral nutrients (e.g., Fe and P), essential to the metabolism of plants and most microorganisms in the soil. LMW organic acids are directly involved in the biogeochem-ical cycles of a variety of metal elements; they serve as pathogenic agents for certain fungi and as wood biodeterioration agents for others; and they have been involved in the detoxification of metal ions in plants, fungi, and bacteria (Dutton and Evans 1996; Jones 1998; Gadd 1999, 2007; Landeweert et al. 2001). Although LMW organic acids are important agents for the mobilization of mineral nutrients from inorganic sources, the strategy most utilized by ECM fungi for the mobilization of N and P from organic sources is enzyme production. Both strategies are used with more or less efficiency depending on the fungal species (Chalot and Brun 1998; Landeweert et al. 2001).

Malate and citrate are the main acids released into the rhizosphere from many plant roots under conditions of iron or phosphorus deficiency. In iron deficiency, it has been suggested that citrate may participate in the dissolution and uptake of Fe3+ by forming Fe3+-citrate complexes in some dicotyledons (Jones 1998). In grasses, phytosiderophores excreted by the roots may be responsible for the solubilization of Fe3+ (Kraemer et al. 2006; Crowley 2006). On the other hand, oxalate, malate, and citrate have been involved in the detoxification of Al in plants through the formation of Al3+-organic acid complexes, which are of lower toxicity for plants than free Al3+ (Jones 1998; Schottelndreier et al. 2001).

LMW organic acids can regulate the speciation and bioavailability of certain metal ions in soils through mobilization by acidification (protonation), formation of soluble complexes (complexolysis), or immobilization by formation of insoluble complexes. These mechanisms are of considerable importance in the acquisition of essential metal ions or metal detoxification by LMW organic acids. The formation of complexes, however, depends not only on the concentration of carboxylate anions, but also on the type and concentration of metal ion, the pH of the medium, and the stability constants of the complexes formed (Jones 1998; Gadd 1999; Meharg 2003). Citrate, malate, and oxalate form stable complexes of high affinity constants with trivalent ions like Fe3+ and Al3+. Oxalate also forms insoluble complexes with Ca2+ and this is one of the main forms in which it is found in soils, fungi, plants, and animals (Jones 1998; Gadd 1999). Despite the high affinities for Fe3+, the stability constants of the complexes with organic acids are inferior to those obtained with hydroxamate or catecholate (Hider 1984; Kraemer 2004), and therefore the role of siderophores in the uptake and transport of Fe3+ must be of higher relevance than chelation by organic acids for living organisms dependent on these metal-chelating agents.

LMW organic acids have been studied in ECM fungi much more than side-rophores, mainly in relation to the weathering of minerals. However, as in the case of siderophores, most detailed studies are limited to a few fungal species. Organic acid exudation by the hyphae of ECM fungi produces the solubilization and mobilization of a variety of nutrients (P, K, Ca, and Mg) directly from solid mineral substrates far from the rhizosphere, insoluble and inaccessible to the roots, thereby contributing to the nutritional status of the host plants (Landeweert et al. 2001; Gadd 2007; van Scholl et al. 2008). The formation of tubular pores in certain minerals has been attributed to organic acid exudation by the hyphae of ECM fungi inside mineral particles, and for this reason they are called rock-eating fungi. In this way, the dissolution of minerals by ECM fungi to obtain essential ions is not limited to an attack on particle surfaces, but inside the mineral particles as well (Jongmans et al. 1997; Landeweert et al. 2001; van Scholl et al. 2008).

The exudation of LMW organic acids, mainly oxalate, by different ECM fungi has been described in vitro in the absence of symbiotic association (Lapeyrie 1988; Lapeyrie et al. 1991; Arvieu et al. 2003; Machuca et al. 2007), in the presence of symbiosis (Ahonen-Jonnarth et al. 2000; Casarin et al. 2003; van Hees et al. 2005, 2006; van Scholl et al. 2006a), and also by ECM root tips harvested in forest ecosystems (Rineau et al. 2008). Some of these studies have shown a greater production of oxalate by mycorrhized compared to nonmycorrhized plants cultivated under axenic conditions (Ahonen-Jonnarth et al. 2000; Casarin et al. 2003). P. involutus, one of the most studied species in relation to oxalate production, can use bicarbonate (NaHCO3) as a source of C and ammonium (NH.4+), or nitrate (NO3~) as N sources to produce the acid and biosynthesis can happen directly from oxaloacetate or via citrate, isocitrate, and glyoxylate (Lapeyrie 1988;

Lapeyrie et al. 1991). In the presence of NO3~, calcium carbonate (CaCO3) and different concentrations of orthophosphate (Pi), P. involutus, H. cylindrosporum, R. roseolus and, S. collinitus released oxalate and protons (H+) to the culture medium (Arvieu et al. 2003). When carboxylates are exuded from the cells, the negative charges must be balanced by a simultaneous efflux of positive charges (like H+), leading to a reduction of pH outside the cells (Jones 1998; Casarin et al. 2003). The increase in the excretion of oxalate and H+ in the presence of CaCO3 suggests the importance that these fungal species may have in the mobilization of nutrients in calcareous soils. In the rhizosphere of the associations between R. roseolus and H. cylindrosporum with P. pinaster, it was possible to detect the release of oxalate in presence of R. roseolus with simultaneous rhizosphere acidification. However, no excretion of oxalate was obtained with H. cylindrosporum and an alkalinization of the rhizosphere was observed (Casarin et al. 2003).

The excretion of citrate and succinate, in addition to oxalate, was detected in a culture medium of R. luteolus, S. verrucosum and S. luteus. S. luteus was the species that produced the highest concentrations of all the organic acids and the only one that also produced malonate. A strong pH reduction was also recorded in the cultures of all the species (Machuca et al. 2007). It must be emphasized that even though these species were not subjected to a nutrient deficit (P, N, or C) and used NH4+ as the source of N, they were able to excrete high organic acid concentrations and H+. Strong acidification was also produced by these species in solid medium in the presence of high concentrations of Cu2+ and Zn2+, but not in the presence of Cd. This suggests that the capacity to produce organic acids and acidify culture media is an intrinsic characteristic of these species. These species were harvested in forest plantations of P. radiata, where the fruiting bodies were found frequently and in large quantities in the different regions studied (Machuca et al. 2007). Van Scholl et al. (2008) indicate that species of the genera Rhizopogon and Suillus are phylo-genetically related to species of the genera belonging to brown rot fungi like Serpula, Coniophora, and Hygrophoropsis, known to produce large amounts of oxalate.

LMW organic acids have also been investigated with respect to the solubiliza-tion of mineral nutrients from a great variety of insoluble substrates and their connection to plant nutrition via ECM fungi. The mobilization of P and Ca from the mineral apatite has been described (Wallander 2000a; Blum et al. 2002; Wallander et al. 2003), as well as K from biotite, microcline, and phlogopite (Paris et al. 1995, 1996; Wallander and Wickman 1999; Wallander 2000b). Using pot experiments, P. sylvestris plants mycorrhized with P. involutus, Piloderma croceum, and H. longicaudum were cultivated with mineral muscovite as the only source of K and hornblende as the source of Mg (van Scholl et al. 2006b). Under these conditions, P. involutus was the only species able to solubilize the muscovite, but not the hornblende; mobilizing K for its host plants. The authors emphasize that ECM fungi can indeed increase the dissolution of minerals in response to a deficiency in nutrients, but the efficiency of the process is clearly species-specific.

The exudation of LMW organic acids has been related to metal ion detoxification mechanisms in ECM fungi (Jentschke and Godbold 2000; Meharg 2003;

Bellion et al. 2006) and plants (Jones 1998). LMW organic acids may act as extracellular chelators, forming complexes with metal ions and preventing them from entering the cell (avoidance mechanisms) in the same way that may occur with siderophores (Jentschke and Godbold 2000; Bellion et al. 2006). In this respect, there is a great deal of controversy, given that when LMW organic acids produce the dissolution of certain minerals in soil, together with increasing the bioavail-ability of essential metal ions, they may also cause the mobilization of potentially toxic metal ions. Furthermore, the efflux of H+ that can occur with the exudation of carboxylate anions may decrease the medium pH, increasing the bioavailability of certain metal ions, making them toxic to fungi and plants (Gadd 2007).

When P. sylvestris seedlings mycorrhized with S. variegatus, R. roseolus, and P. involutus were exposed to high concentrations of metals, a significant increase in oxalate production was observed in the presence of S. variegatus and R. roseolus exposed to Al. In addition, Cu exposure stimulated the production of oxalate in the presence of S. variegatus and P. involutus, and, by contrast, Ni and Cd had no effect on oxalate production (Ahonen-Jonnarth et al. 2000). Using several insoluble minerals, the solubilization capacity was attributed to the strong decrease in extracellular pH caused by different species of ECM fungi (Rosling et al. 2004), and this decrease was also related to metal tolerance (Fomina et al. 2005). Ray and Adholeya (2009) determined in vitro organic acid exudation by P. tinctorius and S. verrucosum using coal ash pond. Formic, malic, and succinic acids were produced by the fungi and a variety of metals (Al, As, Cd, Cr, Ni, and Pb) were detected in their mycelia. No oxalic acid production was described in this paper. Large differences were observed between the strains regarding the patterns of LMW organic acids exuded and metal accumulation. A correlation between organic acid production and a metal accumulation in fungal mycelia was also demonstrated (Ray and Adholeya 2009).

The presence of Ca oxalate crystals has been observed in the cultures of ECM fungi, mycorrhized roots, in the rhizosphere, and around the hyphae of the extraradical mycelium (Cromack et al. 1979; Lapeyrie et al. 1990; Allen et al. 1996; Mahmood et al. 2001; Tuason and Arocena 2009) and it has been suggested that these crystals constitute a reservoir of Ca in the ecosystems, where they can also affect phosphate availability (Dutton and Evans 1996; Gadd 1999, 2007). The formation of these crystals by reprecipitation of solubilized Ca may serve as a fungal detoxification mechanism when the plants grow in soils with high concentrations of Ca; this has also been suggested for other metals that form insoluble oxalates (Cromack et al. 1977; Dutton and Evans 1996; Gadd 2007).

Even though metal tolerance has been extensively researched in ECM fungi, the results with respect to the benefits of the fungi on their host plants can be contradictory at times (Godbold et al. 1998; Jentschke and Godbold 2000; Gadd 2007). This may be related to the lack of experimental evidence clearly showing which mechanisms are involved and how much they contribute to tolerance in fungi. The participation of chelating agents such as siderophores and LMW organic acids has often been suggested among these tolerance mechanisms. However, to date, there seem to be no conclusive studies that are able to correlate the degree of metal tolerance with the type and concentration of chelators produced by an ECM fungus when exposed to high concentrations of one or several metals simultaneously. The situation is of greater complexity when attempting to analyze the fungus-plant system in contaminated soils where the mycorrhized roots might be exposed to more than one metal ion and several other factors that affect the bioavailability of metals. This knowledge is essential if the possible application of certain ECM fungi is to be considered in bioremediation projects or degraded environment reforestation.

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