**3.3 Effect of moment of application on Fe uptake from FeEDDHA components**

#### *Pore water concentrations*

The FeEDDHA component concentrations in the pore water at harvest of pot trial 3 are presented in Figure 10. o,p-FeEDDHA was not detected in any of the samples and has not been included in the figure. In agreement with the results from the other two pot trails, for each of the moments of application separately, racemic o,o-FeEDDHA remained in solution to a larger extent than meso o,o-FeEDDHA. The recovered concentrations only accounted for up to 25% of the racemic o,o-FeEDDHA and up to 8% of the meso o,o-FeEDDHA applied. In particular for the treatment applied at t=6 weeks these low recoveries are remarkable; there was only 2 weeks of residence in the soil-plant system.

For corresponding treatments applied at t=0 and t=3 weeks, the recovery of the treatment applied at t=3 weeks was consequently lower than for the treatment applied at the start of the experiment. This seems counter-intuitive, because the residence time in the soil-plant

Fig. 9. a) Amount of total, racemic and meso o,o-FeEDDHA removed from the soil system per week for the 100%o,o treatment; b) Minimum and maximum FeEDDHA-facilitated Fe uptake (shoot) per week by soybean plants grown on Santomera soil as a function of time, and the amount of racemic o,o-FeEDDHA removed from the soil system per pot per week, both for the 100%o,oL treatment. Error bars have been omitted. (based on Schenkeveld et al., 2010a)

system of the treatment applied at t=0 is 3 weeks longer. An essential difference regarding the system to which the FeEDDHA treatments were applied, is that with application at t=3 weeks, the soybean plants had grown chlorotic and Fe deficiency stress mechanisms had been activated by the time the treatment was applied, whereas plants receiving FeEDDHA treatment at t=0 never grew Fe deficient to this extent in the first place. For strategy I plants like soybean, one of the stress response mechanisms involves up-regulation of the ferric chelates reductase (FCR) system at the root surface (Robinson et al., 1999; Marschner, 1995), enabling plants to more efficiently reduce and take up chelated Fe. Provided that the efficiency of the corresponding EDDHA ligand in complexing and solubilizing Fe from the soil is limited, the FeEDDHA isomer concentration in soil solution will hence decrease more swiftly and strongly in the presence of Fe deficient plants than with plants that are not Fe deficient.

Comparison of corresponding treatments applied at t=0 and t=6 weeks shows that the racemic o,o-FeEDDHA concentrations are comparable (133 and 145 µg l-1), and the meso o,o-FeEDDHA concentrations are approximately twice as high in the t=6 weeks treatment (20 and 47 µg l-1); still these differences in meso o,o-FeEDDHA concentration are small in comparison to the dosage applied (560 µg l-1). The effect of stress response mechanisms on o,o-FeEDDHA concentrations equaled six weeks of residence time in the soil-plant system for racemic o,o-FeEDDHA, and over 3 weeks for meso o,o-FeEDDHA.

#### *Fe uptake*

The Fe uptake data presented in Figure 11 demonstrate that, in agreement with Rojas et al., 2008, o,p-FeEDDHA did not significantly increase Fe uptake in any of the treatments; neither applied as a single substance (*o,p 3* and *o,p 6* treatment) nor in a mixture through a

Fig. 9. a) Amount of total, racemic and meso o,o-FeEDDHA removed from the soil system per week for the 100%o,o treatment; b) Minimum and maximum FeEDDHA-facilitated Fe uptake (shoot) per week by soybean plants grown on Santomera soil as a function of time, and the amount of racemic o,o-FeEDDHA removed from the soil system per pot per week, both for the 100%o,oL treatment. Error bars have been omitted. (based on Schenkeveld et al.,

system of the treatment applied at t=0 is 3 weeks longer. An essential difference regarding the system to which the FeEDDHA treatments were applied, is that with application at t=3 weeks, the soybean plants had grown chlorotic and Fe deficiency stress mechanisms had been activated by the time the treatment was applied, whereas plants receiving FeEDDHA treatment at t=0 never grew Fe deficient to this extent in the first place. For strategy I plants like soybean, one of the stress response mechanisms involves up-regulation of the ferric chelates reductase (FCR) system at the root surface (Robinson et al., 1999; Marschner, 1995), enabling plants to more efficiently reduce and take up chelated Fe. Provided that the efficiency of the corresponding EDDHA ligand in complexing and solubilizing Fe from the soil is limited, the FeEDDHA isomer concentration in soil solution will hence decrease more swiftly and strongly in the presence of Fe deficient plants than with plants that are not

Comparison of corresponding treatments applied at t=0 and t=6 weeks shows that the racemic o,o-FeEDDHA concentrations are comparable (133 and 145 µg l-1), and the meso o,o-FeEDDHA concentrations are approximately twice as high in the t=6 weeks treatment (20 and 47 µg l-1); still these differences in meso o,o-FeEDDHA concentration are small in comparison to the dosage applied (560 µg l-1). The effect of stress response mechanisms on o,o-FeEDDHA concentrations equaled six weeks of residence time in the soil-plant system

The Fe uptake data presented in Figure 11 demonstrate that, in agreement with Rojas et al., 2008, o,p-FeEDDHA did not significantly increase Fe uptake in any of the treatments; neither applied as a single substance (*o,p 3* and *o,p 6* treatment) nor in a mixture through a

for racemic o,o-FeEDDHA, and over 3 weeks for meso o,o-FeEDDHA.

2010a)

Fe deficient.

*Fe uptake* 

Fig. 10. Racemic and meso o,o-FeEDDHA concentration in the pore water of Santomera soil at harvest. Error bars indicate standard deviations. Letters indicate the significantly different groups as identified by the Tukey post-hoc test, for both FeEDDHA components separately. (based on Schenkeveld et al., 2010b)

synergistic effect (*o,o-mix low + o,p*). Due to its interaction with soil constituents, the residence time of o,p-FeEDDHA in soil solution can be short (Schenkeveld et al., 2007). Therefore o,p-FEDDHA had only been applied to soybean plants that were already Fe deficient at the moment of application. However, even when applied in the growth stages that Fe requirements were highest and Fe stress response mechanisms were activated, facilitating a more efficient Fe uptake, o,p-FeEDDHA still did not significantly increase the Fe uptake of soybean plants.

Both racemic o,o-FeEDDHA and meso o,o-FeEDDHA did contribute to Fe uptake (Figure 11), as shown from the fact that in all treatments with o,o-FeEDDHA, Fe uptake was significantly higher than in the blank treatment. This is in agreement with the conclusion from the study by Ryskievich and Boku (1962). For none of the moments of application, significant differences in Fe uptake were found between the *racemic o,o* and *meso o,o* treatment. Because overall Fe uptake in the *o,o-mix high* treatments was higher than in the *racemic o,o* (p = 0.030) and the *meso o,o* (p = 0.012) treatments, Fe uptake was not yet maximal in the *racemic o,o* and the *meso o,o* treatments. Therefore it can be concluded that racemic and meso o,o-FeEDDHA were approximately equally effective in facilitating Fe uptake. Lucena and Chaney (2006) reported that meso o,o-FeEDDHA was more effective in delivering Fe to hydroponically grown cucumber plants than racemic o,o-FeEDDHA, as a result of a lower stability favouring Fe reduction at the root surface. Possibly in soil, a preferential Fe uptake from meso o,o-FeEDDHA was balanced by a higher affinity for the solid phase and a faster decline in soil solution concentration.

Moreover, for both the *racemic o,o* and *meso o,o* treatments, no significant difference in Fe uptake was observed between the different moments of application. This is remarkable, because the plants receiving treatment after 6 weeks had much less time to benefit from the applied o,o-FeEDDHA. Apparently, as a result of Fe deficiency stress response mechanisms and development of the root system, the soybean plants had grown much more efficient with regard to Fe uptake. In only two weeks time, the soybean plants from the *racemic o,o 6* and *meso o,o 6* treatments took up an additional 0.36 mg of Fe per pot, which corresponds with 50% of the total Fe uptake in the blank treatment.

Fig. 11. Fe uptake by soybean plants grown on Santomera soil for all FeEDDHA treatments. Error bars indicate standard deviations. Letters indicate the significantly different groups as identified by the Tukey post-hoc test including all FeEDDHA treatments and all moments of application. (based on Schenkeveld et al., 2010b)
