1. A Comprehensive Mössbauer Study of the Iron Uptake Strategies in Plants K. Kovács1, E. Kuzmann1, F. Fodor 2, A. Vértes1 1Laboratory of Nuclear Chemistry, Chemical Institute, Eötvös Loránd University, Pázmány P. s. 1/a, Budapest 1117, Hungary 2Department of Plant Physiology and Molecular Plant Biology, Eötvös Loránd University, Pázmány P. s. 1/c, Budapest 1117, Hungary

2. Major processes to be involved in metal accumulation by plants
The absorption and storage of metal cations in plants is of great importance for plant physiology and phytoremediation-based biotechnology. The mechanism of iron uptake in plants has considerable scientific and technological interest due to the need for improved crop production. Plants use two distinct strategies to solubilize and absorb iron from soil. They either reduce Fe(III) complexes at the root surface and absorb the resulting Fe2+ ions produced via root-associated reduction (strategy group I), or excrete specific Fe(III)-binding, low-molecular-weight organic polydentate ligands known as phyto-siderophores, which solubilize Fe3+ ions and make them available for absorption (strategy group II). These two strategies are used by two distinctly separate taxonomic groups of plants, with the phytosiderophore-based mechanism restricted to the grass family and the Fe(III) reduction mechanism restricted to the dicots and monocots not included in the grass family. In our work two plants were used to investigate the different iron uptake mechanisms, namely cucumber (Cucumis sativus) which belongs to the strategy group I. and wheat (Triticum aestivum), a member of the strategy group II.
Major processes to be involved in metal accumulation by plants
Distribution and sequestration
Xylem transport
Absorption and translocation
Root-microbe interaction

3. According to the literature data, Mössbauer spectroscopy was applied to investigate iron in duckweed, stocks, soyabean and pea (Goodman and Dekock, 1982, Goodman et al., 1982). According to these results, all specimens showed the presence of iron in the ferric form. Besides most of the iron was identified as ferritin, though a small fraction (< 15%) present in complexes could not be discounted. A detailed study of iron uptake in rice (Kilcoyne et al., 2000) showed mainly different Fe(III)-oxide components precipitated in the root cell walls. The spectra were also found to be characteristic of Fe(III) in ferritin and other complexed forms that could not be further identified by the authors. Iron biomineralization was observed in a perennial grass, isolated from extreme acidic environment with high contents of metal where the Mössbauer spectral analysis indicated that iron accumulated in this plant mainly as jarosite and ferritin (Rodríguez et al., 2005).
For details, see the following references:
Goodman, B.A., DeKock, P.C. (1982) J. Plant Nutr. 5,
Goodman, B.A., DeKock, P.C., Rush, J.D. (1982) J. Plant Nutr. 5,
Kilcoyne, S.H., Bentley, P.M., Thongbai, P., Gordon, D.C., Goodman, B.A. (2000) Nucl. Instrum. Meth. Phys. Res. B. 160,
Rodríguez, N., Menéndez, N., Tornero, J. Amils, R., de la Fuente (2005) New Phytologist, 165,
The aim of our work was to apply 57Fe Mössbauer spectroscopy to get information about the valence state and microenvironments of iron taken up by the root of cucumber and wheat via their responses to iron-deficiency, to check the iron uptake strategies, to study the iron distribution in the root tissue. The experimental conditions were the following:
Plants were grown in iron sufficient (10-5 M 57Fe-citrate) and iron deficient (no iron added) nutrient solutions. Iron deficient plants were kept in iron containing solutions for different time intervals (5∙10-4 M 57Fe-citrate, 30 min – 24 hours) or for 30 min using different iron concentrations (10-5 M – 5∙10-4 M 57Fe-citrate). Mössbauer spectra of the roots were taken at liquid nitrogene temperature. .

4. FeIIIA FeIIIB FeIIIC  / (mms-1) 0,50 EQ / (mms-1) 0,44 0,80 1,21
Mössbauer spectra of iron sufficient roots (grown in 10-5 M 57Fe-citrate containing solution) with the corresponding Mössbauer parameters:
FeIIIA
FeIIIB
FeIIIC
 / (mms-1)
0,50
EQ / (mms-1)
0,44
0,80
1,21
A / % 44 35 21
cucumber
FeIIIA
FeIIIB
FeIIIC
 / (mms-1)
0,51
0,48
EQ / (mms-1)
0,75
1,15
A / % 39 41 21
wheat

5. Mössbauer spectra of iron deficient roots which were kept in iron containig solutions (5∙10-4 M 57Fe-citrate) for different time intervals (shown in the figures)
CUCUMBER
WHEAT

6. The Mössbauer spectrum recorded at T=80 K of the iron sufficient roots can be decomposed into three symmetrical doublets. These doublets can be associated with three different FeIII microenvironments. The Mössbauer parameters calculated show that the major component in the root is a high spin iron(III) in octahedral coordination (FeIIIA) possibly resulting from iron(III)-carboxylate complexes. These carboxylate complexes are suggested to be located mainly inside the cell since they can not be removed with any of the chemical washing procedures applied to mobilize cell-wall bound iron. The Mössbauer parameters of the FeIIIB component are in good agreement with those reported for iron oxyhydroxides or hydrous ferric oxides. Moreover, this quadrupole doublet is similar to those reported for the ferritin found in several plant tissues. The third doublet (FeIIIC) with the highest quadrupole splitting represents a sulfate/hydroxide containing FeIII species. According to its Mössbauer parameters, it can be attributed to jarosite or its analogous compound. The jarosite is suggested to be present in the cell wall since the nutrient solution contained enough sulfate and potassium.
The Mössbauer spectra of iron deficient cucumber roots supplied with 0.5 mM Fe-citrate for 30 min can be decomposed similar to the iron sufficient samples but in this case, an FeII component (ferrous hexaaqua complex) can also be found beside the FeIIIA, FeIIIC component as it is shown in the figure. The relative amount of the different iron species depend on the time period of iron supply: the FeII shows a significant decrease (from 48% to 6%) with the iron treatment, while the relative amount of the main iron component, FeIIIA increases from 32% to 91% after 24 hours and the FeIIIC component decreases from 20% to 3%.

7. FeIII FeII FeII FeIII FeII membrane
The changes in the relative content of the components shown in the figure can indicate the transformation of FeII-hexaaqua complex into the FeIII species. This agrees with the accepted viewpoint that the translocation and storage of iron inside the root cells take place in the form of iron(III) compounds. The reaction rate of this FeII-to-FeIII transformation is much higher than that of the FeIII reduction outside the membrane, when sufficient amounts of iron are taken up by the root, possibly due to significantly lower FeII reduction rate in these plants. Consequently, the FeII species cannot be detected on the background of the FeIII components using Mössbauer spectroscopy in the case of iron-sufficient roots.

8. Mössbauer spectra of iron deficient roots which were kept in iron containig nutrient solutions for 30 min. Iron concentration was varied according to the figure:
Iron content of the roots measured with ICP-MS:
10-5 M
10-4 M
5∙10-5 M
5∙10-4 M
The relatively low amount of FeII in the case of the lowest applied 57FeIII-citrate concentration can be explained by the very fast uptake and reoxidation process. Comparing the total iron concentration in the root with the amount reducible in 30 min we can say that the reduction rate is high enough to potentially reduce all Fe accumulated outside the cell. This is in agreement with the data obtained with Fe sufficient plants with the background of enhanced reducing capacity under Fe deficiency (data not shown). This may result in the accumulation of FeII outside the plasma membrane

9. In conclusion, we have succeeded in showing by Mössbauer spectroscopy the presence of divalent iron in the plant root when the nutrient solution contained only FeIII. This gives a direct evidence for the existence of Fe2+ ions produced via root-associated reduction according to the mechanism proposed for iron uptake in plants belonging to strategy group I as in the case of cucumber. Moreover, the results obtained for the wheat, a model plant for strategy II iron uptake where no iron(II) was found, support the iron-uptake mechanism suggested in the case of strategy group II.
We have shown that the iron-reduction and iron-transport in the case of the strategy group I depends strongly on the time and the concentration of the iron supply after iron deficiency. We suppose that the limiting step in the iron-uptake is the iron transport through the membranes after the reduction which can be significantly enhanced in iron deficiency.
Data obtained in the iron-sufficient cases show that iron is distributed among three characterisitic main species in the roots of different plants under nutrition condition sufficient for iron. They can be asssociated with jarosite related to the cell wall and ferritin as well as some FeIII-carboxylate compounds located mainly inside the plant cell. The similarity found in the iron storage of cucumber and that of wheat can lead to a more general statement that iron is stored in a very similar form in the roots of different plants even belonging to different strategy groups.
K. Kovács, E. Kuzmann, F. Fodor, A. Vértes, A. A. Kamnev, Hyp. Int. 165 (2005)