Iron Uptake From Iron Chelates

When an Fe chelate is present in the soil solution, it will refill the Fe pool in the root surface as soon as the plant uses it.

resulting in:

Therefore, the higher the K° and the concentration of free ligand, the lower the concentration of Fe3+ allowed by the chelate to be available for the plant. This Fe3+ should be reduced before entering the plant. In Strategy I plants the reduction of the Fe-chelate is carried out by an Fe-chelate reductase (FCR) enzyme (Chaney et al., 1972; Bienfait, 1985; Schmidt, 2005), whereas in Strategy II plants (Kawai and Alam, 2005) chelating agents would compete with phytosiderophores for Fe binding. Since Fe-phytosiderophore complexes are far less stable than synthetic chelates, this may be a problem for the use of Fe-chelates by these plants. After the normal or FCR mediated reduction, Fe2+ transport into roots occurs by a plasma membrane transporter (Fox et al., 1996).

Figure 5-9. Comparison of the Fe reduced by green stressed cucumber plants at pH 7.5 from different Fe-chelates and the concentration of Fe in shoots when the same chelates were used at low concentrations (5 ^M) in hydroponic culture (pH 7.5). Chelates are presented in stability sequence (from lower to higher: A: EDTA/Fe3+; B: PDDHA/Fe3+ (pentil diamine di(o-hydroxyphenilacetic) acid; C: meso o,o- EDDHA/Fe3+; D: o,o-EDDHA/Fe ; E: o,o-EDDHMA/Fe3+; F: rac o,o-EDDHA/Fe3+; G: EDDHSA/Fe3+; and H: HBED/Fe3+). Data re-elaborated after Lucena and Chaney, 2005a and 2005b.

Figure 5-9. Comparison of the Fe reduced by green stressed cucumber plants at pH 7.5 from different Fe-chelates and the concentration of Fe in shoots when the same chelates were used at low concentrations (5 ^M) in hydroponic culture (pH 7.5). Chelates are presented in stability sequence (from lower to higher: A: EDTA/Fe3+; B: PDDHA/Fe3+ (pentil diamine di(o-hydroxyphenilacetic) acid; C: meso o,o- EDDHA/Fe3+; D: o,o-EDDHA/Fe ; E: o,o-EDDHMA/Fe3+; F: rac o,o-EDDHA/Fe3+; G: EDDHSA/Fe3+; and H: HBED/Fe3+). Data re-elaborated after Lucena and Chaney, 2005a and 2005b.

The FCR enzyme is up-regulated by Strategy I plants when Fe is limited in the media (Robinson et al., 1999). The substrates of the FCR are stable, Fe(III)-selective chelates. Chaney (1989) studied the effect of Fe-chelate concentration and nature on the rate of Fe reduction by roots of Fe-deficient peanut plants. No conclusive relation between kinetic parameters (the rate of Fe reduction by roots) and the stability of the Fe-chelates was observed using EDDA (ethylene-diamine-diacetic acid), HEDTA, EDTA and DTPA as chelators. The charge of the species in solution, which varied from 0 to -2 among the four chelates used, could have controlled the rate of reduction. The more stable Fe-chelates were not used in those experiments. FCR has been described to have higher activities at pH values below 6.5 (Moog and Bruggemann, 1995; Susin et al., 1996; Romera et al., 1998), while Fe chlorosis occurs in calcareous soils with bulk soil pH 7.5-8.5. Strategy I also involves up-regulation of H+ extrusion (Romheld and Marshner, 1986;

Alcántara, 1991; Wei et al., 1997) that may help in a local reduction of the rhizosphere or apoplastic pH, despite the high buffering effect of bicarbonate (Lucena et al., 2000).

Recently, the role of the Fe3+-chelates as substrates of the FCR enzyme, a part of the Fe uptake mechanism in Strategy I plants, has been investigated (Lucena and Chaney, 2005a). It was concluded that a high stability of the Fe-chelate decreases FCR reduction rates in mildly chlorotic cucumber plants, although plants can always take up Fe from very stable Fe-chelates (see also Chaney, 1988). Also, a low Fe concentration in xylem sap was found in plants treated with very stable Fe-chelates. In another experiment (Lucena and Chaney 2005b), cucumber plants were grown with 5 ^M concentrations of different chelates. Various Fe nutrition indexes revealed that plants had a better Fe supply when treated with relatively weaker chelates, with the exception of EDTA/Fe3+. A comparison of the results from both experiments is presented in Figure 5-9, showing a similar trend for both parameters in all chelates, excepting EDTA/Fe3+. For chelates other than EDTA/Fe3+, Fe uptake correlates with Fe reduction. Considering the chemical reaction:

where Kred is the stability constant for the reduction of the Fe3+ to Fe2+ (log Kred = 13.04, Lindsay, 1979) and K°s is the stability constant of the formation of the Fe(III) chelate.

The presence of Fe2+ will be therefore controlled by three terms: the first one depends inversely on the stability constant of the Fe(III) complex; the second one depends on the free chelating agent concentration, which is controlled by some side-reactions (protonation and complexation with other metals); and the third is the redox potential that the plant FCR enzyme imposes on the system. Then, the weaker the Fe-chelate, the easier plants can reduce Fe. Although this theoretical scenario could be too simplistic, it is in good agreement with the obtained results (Figure 5-9), where chelates are presented in a sequence from lower (left) to higher (right) stability. We also observed the same behaviour comparing o,o- EDDHA/Fe3+ and op-EDDHA/Fe3+ While o,o-EDDHA/Fe3+ is more stable than op-EDDHA/Fe3+, the latter can provide Fe faster to the plant (Garcia-Marco et al., 2005).

The EDTA/Fe2+ complex is also quite stable, and therefore the Fe2+ produced by the FCR activity can be trapped again by free EDTA, competing with BPDS present in the Fe reduction assays. In fact, FCR activities could be twice (at pH 6.0) or 3.5 times (at pH 7.5) faster when EDTA/Fe3+ is the substrate than when the source is o,o-EDDHA/Fe3+. However, the Fe concentration in the xylem sap is double for o,o-EDDHA/Fe3+ than for EDTA/Fe3+, which is in good agreement with the better Fe nutrition provided by o,o- EDDHA/Fe3+ (Figure 5-9).

The determination of the amount of Fe reduced by plants supplied with different Fe-chelates can inform us only about a part of the Fe uptake process. The result of the complete process could be better evaluated from the Fe absorption by the plant. Chaney and Bell (1987) presented data of the Fe reduction from Fe-DTPA and the 59Fe-uptake by stressed peanut plants. In that study, the fraction of Fe reaching the shoots was quite large, close to 100% of the reduced Fe, but the pH values of the uptake assay solutions were approximately 4. The authors observed separate regulation of Fe reduction and uptake rates, with acidification being one of the factors related with the differences in regulation. In our studies (Lucena and Chaney, 2005a and 2005b) Fe uptake was calculated from the concentration of Fe present in the xylem sap, and in both experiments only a small fraction (around 1.1% at pH 6.0 and 0.6% at pH 7.5) of the Fe reduced reached the xylem sap. Discrepancies with the data of Chaney and Bell (1987) may be due by the effect of pH, since we used higher pH values (6.0 or 7.5) than that employed by Chaney and Bell (1987) (around 4.0). Moreover, Chaney and Bell (1987) did not use a pH buffer in the uptake solutions, so that acidification by the roots could likely have increased both the reduction and uptake processes, while in our experiments (Lucena and Chaney, 2005a and 2005b) the final pH was close to the initial one, plant-driven acidification being prevented from affecting both uptake and reduction of Fe.

The Fe2+ produced by the FCR that was not absorbed by the plant had to be re- oxidised (or complexed in the case of EDTA). The ultimate electron acceptor for the re-oxidation should be oxygen, but the chelating agents could also catalyse the re-oxidation by favouring the formation of Fe3+-chelate, or could even act as electron carriers (Kurimura et al., 1968). The role of chelating agents in Fe2+ re-oxidation under natural conditions of plant growth should receive more consideration, since it may affect the "shuttle effect" that has been proposed for the action of Fe-chelates in soils (Lindsay, 1995).

While Fe reduction and uptake measured in a controlled experiment gave us important information on the factors affecting the efficacy of Fe chelates, biological experiments including the soil are also needed to have a better knowledge of the effect of Fe-chelates. Several authors have made experiments to compare different chelates (Reed et al., 1988; Hernandez-Apaolaza et al., 1995; Alvarez-Fernandez et al., 1996), mainly o,o- EDDHA/Fe3+ and EDDHMA/Fe3+, but they have obtained contradictory results, because commercial chelates with unknown Fe-chelate content were used. Recently (Alvarez-Fernandez et al., 2005) we were able for the first time to compare chelates with different chelating agents using the same amount of chelated Fe, since a reliable determination method is now available (Lucena et al., 1996 and Hernandez-Apaolaza et al., 1997). Alvarez-Fernandez et al. (2005) compared the application of chelates with the same doses of chelated Fe in three different experiments. We concluded that commercial products containing o,o- EDDHA/Fe3+ and EDDHMA/Fe3+ had a similar efficacy to re-green Fe chlorotic plants, both in a soil-less system and in field conditions. However, o,o-EDDHA/Fe3+ presented some advantages, due to its somewhat longer lasting effect. EDDHSA/Fe3+ was also effective in correcting Fe chlorosis in pear trees grown on a calcareous soil and in sunflower plants grown in a soil-less system.

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