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
E-mail: kkriszti@bolyai.elte.hu
http://www.chem.elte.hu/departments/magkem/hun/index.html
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 phytosiderophores, 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
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, 345-353.
Goodman, B.A., DeKock, P.C., Rush, J.D. (1982) J. Plant Nutr. 5, 355-362.
Kilcoyne, S.H., Bentley, P.M., Thongbai, P., Gordon, D.C., Goodman, B.A. (2000) Nucl. Instrum.
Meth. Phys. Res. B. 160, 157-166.
Rodríguez, N., Menéndez, N., Tornero, J. Amils, R., de la Fuente (2005) New Phytologist, 165,
781-789.
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.
.
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
0,50
0,50
E Q /
0,44
0,80
1,21
44
35
21
FeIIIA
FeIIIB
FeIIIC
 / (mms-1) 0,51
0,48
0,48
E Q /
0,48
0,75
1,15
39
41
21
1,003
relative transmission
0,970
0,936
0,903
0,870
cucumber
0,836
(mms-1)
0,803
0,769
-8
-6
-4
-2
0
2
4
6
8
-1
A/%
v / (mm s )
relative transmission
1,000
0,998
0,996
wheat
(mms-1)
0,994
-6
-4
-2
0
2
-1
v / (mm s )
4
6
A/%
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
1,000
1,000
0,995
30 min
0,990
30 min
relative transmission
relative transmission
1,000
60 min
0,995
1,000
0,995
90 min
0,990
1,000
0,980
1,000
180 min
0,995
1,000
180 min
0,990
1,000
0,990
24 hours
24 hours
0,980
-6
-4
-2
0
2
-1
v / (mm s )
4
6
-6
-4
-2
0
2
-1
v / (mms )
4
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%.
membrane
FeIII
relative occurrence
II
of Fe , %
FeII
45
II
30
Fe
FeII
15
FeII
FeIII
relative occurrence
III
of Fe C, %
relative occurrence
III
of Fe A, %
100
0
80
60
III
Fe
A
40
20
15
III
10
Fe
C
5
0
0
4
8
12 16 20 24
t, h
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-toFeIII 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.
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:
2000
 g Fe / g dry weigth
1,000
0,999
relative transmission
10-5
M
1,000
total iron content
iron content inside the cell
2+
content of Fe
1500
1000
500
0
0,0
0,998
-4
1,0x10
-4
2,0x10
-4
3,0x10
-4
4,0x10
-4
5,0x10
cFe-citrate / M
5∙10-5
0,996
M
1,000
0,995
10-4 M
1,000
0,995
5∙10-4 M
-6
-4
-2
0
2
-1
v / (mm s )
4
6
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
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) 289-294