**3.2 FeEDDHA-facilitated Fe uptake as a function of time**

#### *Pore water concentrations*

96 Soybean Physiology and Biochemistry

0.0 0.5 1.0

Fig. 6. a) Fe uptake; b) Fe content of the shoot; and c) dry weight yield (shoot) of soybean plants grown on Santomera soil as a function of the o,o-FeEDDHA content of the FeEDDHA treatment. Error bars indicate standard deviations. (based on Schenkeveld et al., 2008)

**o,o-FeEDDHA fraction**

b

c

10

30

**Fe content shoot (mg kg(dw)-1)**

50

70

In Figure 7, the Fe and FeEDDHA component concentrations are presented as a function of time for the 30%o,oL treatment from pot trial 2. Within the first week of the experiment, the Fe concentration underwent a strong drop, from 4.25 to 0.81 mg l-1 Fe, after which it gradually declined further (Figure 7a). This drop was largely caused by the practically complete removal of o,p-FeEDDHA and rest-FeEDDHA from soil solution. From 1 week onward, the Fe concentration was largely (> 92%) governed by racemic and meso o,o-FeEDDHA (Figure 7b). The concentration of racemic and meso o,o-FeEDDHA underwent two stages: 1) a rapid, strong decline within the first week, and 2) a gradual decline from one week onward. The initial decrease in racemic o,o-FeEDDHA concentration (≈28%) was smaller than for meso o,o-FeEDDHA (≈54%). This fast decline has been attributed to adsorption, which can be described with linear adsorption isotherms (Schenkeveld et al 2010a). The rate of the gradual decline was higher for meso o,o-FeEDDHA than for racemic o,o-FeEDDHA, resulting in a continuous increase in relative contribution of racemic o,o-FeEDDHA to the total Fe in solution. The nature of the gradual decline differed for racemic and meso o,o-FeEDDHA: for meso o,o-FeEDDHA it could be accurately described with an exponential decay function, whereas for racemic o,o-FeEDDHA no decline was observed in the second week of the experiment and from 2 weeks onward, the rate of decline was less consistent (Figure 7b). The decay constant in the exponential function describing the gradual decline in meso o,o-FeEDDHA concentration proved dependent on the applied amount of meso o,o-FeEDDHA (Schenkeveld et al., 2010a).

#### *Fe uptake*

Fe uptake as a function of time is presented in Figure 8 and was calculated by subtracting total Fe uptake of two consecutive harvesting rounds for a corresponding treatment. Fe uptake at 2 weeks actually represent the Fe taken up during the second week, and so on. During the 2nd week, in the early vegetative stage, Fe requirements were still low. Chlorosis had just developed in the soybean plants and possibly utilization of Fe which had been present in the seeds, still covered part of the Fe requirements. In the 3rd and the 4th week, during the progressed vegetative stage, Fe demand strongly increased and in the blank treatment chlorosis was most severe. In the course of the 4th and during the 5th week, the transfer from the vegetative to the reproductive stage took place; the plants flowered and started to grow pods. In the 6th week, the seed formation inside the pods progressed and Fe requirements were even larger than during the vegetative stage, in order to provide the seeds with sufficient Fe (Grusak, 1995). Throughout the experiment, the sequence in Fe uptake was: blank < 30%o,o < 100%o,o. The difference in Fe uptake among the treatments was largest in growth stages in which Fe requirements were largest. The large differences in Fe uptake during the reproductive stage did not show in an increased difference in SPADindices (Schenkeveld et al., 2010a).

#### *Relation between FeEDDHA removal and Fe uptake*

The amount of FeEDDHA components removed from the soil system (solid and solution phase combined) per week was calculated from the decrease in soil solution concentration (Figure 8b), assuming linear adsorption (Schenkeveld et al., 2010a), and is presented as a function of time for the 100%o,o treatment in Figure 9a. The removal of meso o,o-FeEDDHA was larger than the removal of racemic o,o-FeEDDHA throughout the experiment. Still, racemic o,o-FeEDDHA, seems to have a more pronounced influence on the shape of the total o,o-FeEDDHA removal-curve (Figure 9a).

Fig. 7. Fe and FeEDDHA component concentrations in the pore water of Santomera soil as a function of time for the 30%o,o treatment. Error bars indicate standard deviations. (based on Schenkeveld et al., 2010a)

01234567

**time (weeks)**

Fig. 7. Fe and FeEDDHA component concentrations in the pore water of Santomera soil as a function of time for the 30%o,o treatment. Error bars indicate standard deviations. (based on

Schenkeveld et al., 2010a)

0

**a**

1

2

**[Fe] (mg l-1**

 **Fe)**

3

4

Fig. 8. Fe uptake (shoot) by soybean plants grown on Santomera soil as a function of time. Error bars have been omitted. (based on Schenkeveld et al., 2010a)

In Figure 9b, two scenarios for FeEDDHA-facilitated Fe uptake are presented as a function of time for the 100%o,o treatment. In the maximum FeEDDHA-facilitated uptake scenario, all Fe uptake by the soybean plants is assumed FeEDDHA-facilitated; in the minimum FeEDDHAfacilitated uptake scenario, only the Fe uptake in addition to Fe uptake in the blank treatment is assumed FeEDDHA-facilitated. The shape of the racemic o,o-FeEDDHA removal curve strongly resembles the shape of the FeEDDHA-facilitated Fe uptake curves (Figure 9b). This suggests that the removal of racemic o,o-FeEDDHA from the soil system is to a large extent plant-related. The fact that the gradual decline in racemic o,o-FeEDDHA concentration only started after 2 weeks, when the plants developed a strong need for Fe, further supports this reasoning. The shape of the meso o,o-FeEDDHA removal curve (Figure 9a) does not show a similar resemblance, which suggests that the removal of meso o,o-FeEDDHA from the soil system is to a large extent non-plant related. The nature of the plant-independent process causing a decline in meso o,o-FeEDDHA concentration remains unclear.
