Plant Cell Physiol. 42(1): 94–105 (2001)
JSPP © 2001
Technical Advance: Reduction of Fe(III)-Chelates by Mesophyll Leaf Disks of
Sugar Beet. Multi-Component Origin and Effects of Fe Deficiency
Ajmi Larbi 1, Fermín Morales 1, Ana Flor López-Millán 1, Yolanda Gogorcena 1, Anunciación Abadía 1,
Petra R. Moog 2 and Javier Abadía 1, 3
1
2
Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, C.S.I.C., Apdo. 202, E-50080 Zaragoza, Spain
Botanisches Institut, Johann Wolfgang Goethe-Universität, Senckenberganlage 31-33, D-60054 Frankfurt/Main, Germany
;
The characteristics of the Fe(III)-chelate reductase
activity have been investigated in mesophyll disks of Fesufficient and Fe-deficient sugar beet leaves. The Fe(III)chelate reductase activity of mesophyll disks was light
dependent and increased markedly when the epidermis was
removed. Iron(III)-citrate was photo-reduced directly by
light in the absence of plant tissue. Total reductase activity
was the sum of enzymatic mesophyll reduction, enzymatic
reduction carried out by organelles exposed at the disk edge
and reduction caused by the release of substances both by
exposed mesophyll cells and at the disk edge. Compounds
excreted were shown by HPLC to include organic anions,
mainly oxalate, citrate and malate. When expressed on a
leaf surface basis, Fe deficiency decreased the total mesophyll Fe(III)-chelate reductase activity. However, Fe-sufficient disks reduced less Fe than the Fe-deficient ones when
expressed on a chlorophyll basis. The optimal pH values for
Fe(III) reduction were always in the range 6.0–6.7. In control leaves Fe(III)-citrate and Fe(III)-malate were the substrates that led to the highest Fe reduction rates. In Fe-deficient leaves Fe(III)-malate led to the highest Fe reduction
rates, followed by Fe(III)-EDTA and then Fe(III)-citrate.
Km values for the total reductase activity, enzymatic mesophyll reduction and enzymatic reduction carried out by
organelles at the disk edge were obtained.
acquisition by plant roots was recognized since the original
work by Brown and co-workers (Chaney et al. 1972), which
showed the existence of an obligatory Fe reduction step from
Fe(III) to Fe(II) prior to uptake in the roots of dicotyledonous
plants. The increase in the capacity to reduce Fe(III) is
considered an integral part of the so-called Strategy I, that
involves a number of mechanisms resulting in an improvement
in Fe acquisition in Fe-deficient dicots and non-Poaceae
monocots (Marschner et al. 1986, Welkie and Miller 1993,
Marschner and Römheld 1994). The increase in the capacity to
reduce Fe(III) in Fe-deficient plants is thought to be caused
by one or several plasma membrane (PM)-bound FC-R
enzyme(s) (Bienfait 1985, Cakmak et al. 1987, Bienfait 1988).
These FC-R enzymes (Rubinstein and Luster 1993, Moog and
Brüggemann 1995) would reduce chelated Fe(III) present in
the root/soil interface to Fe(II) which then can be transported
into the root.
However, Fe acquisition from the soil phase is not the
only limiting step in Fe utilization by plants. After plants
acquire Fe it must be transported to its sites of utilization all
over the plant. Since most (up to 80%) of the leaf Fe is located
in the chloroplast, to arrive at its final destination most of the
Fe must cross several biological membrane systems. The characteristics of this multi-step internal transport system are still
largely unknown, although there is evidence in the literature
suggesting that some steps in the internal transport system may
be impaired by Fe deficiency itself. For instance, it is well
known that in many cases leaves from field-grown Fe-deficient plants have total Fe concentrations similar to those of
control, Fe-sufficient leaves (Abadía 1992). This may suggest
that, when Fe-deficient, part of the Fe acquired from the soil by
the FC-R could be immobilized and accumulated in inactive
forms somewhere in the leaf (Morales et al. 1998). Therefore,
more information is needed on the fine characteristics of the
different steps in the Fe transport systems, and also on the
changes induced in those systems by Fe deficiency.
Few data are available in the literature on how Fe enters
the leaf cell. Iron is commonly assumed to be transported in the
xylem as Fe(III)-citrate complexes (Tiffin 1966), and has been
proposed to be reduced at the leaf cell PM by a FC-R similar to
that of the root PM (Moog and Brüggemann 1995). The existence of a mesophyll FC-R related to Fe uptake and capable of
Key words: Beta vulgaris — Iron deficiency — Iron reduction
— Mesophyll — Sugar beet.
Abbreviations: FC-R, ferric chelate-reductase; PM, plasma
membrane; PDTS, 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4triazine; PPFD, photosynthetic photon flux density; Rec and Rem, Fe
reduction due to enzymatic processes at the cut edge and at the mesophyll cell plasma membrane, respectively; Rlc and Rlm, Fe reduction
due to compounds released from the leaf disk cut edge and undamaged exposed mesophyll cells, respectively; SPAD, portable leaf Chl
meter; Tr, total Fe reduction by leaf disks.
Introduction
The importance of a ferric chelate reductase (FC-R) in Fe
3
Corresponding author: E-mail, [email protected]; Fax, +34 976 716145.
94
Leaf Fe(III) reduction and Fe deficiency
using Fe(III)-EDTA and Fe(III)-citrate as substrates was first
demonstrated in Fe-sufficient and deficient leaves of Vigna
unguiculata (Brüggemann et al. 1993). De
de la Guardia and
Alcántara (1996) confirmed the presence of a mesophyll FC-R,
reducing Fe(III)-EDTA, in Helianthus annuus. In the latter
case, it was shown with vacuum-infiltrated leaf disks that Fe
deficiency did not change the activity of the FC-R on a leaf
area basis, although the biochemical characteristics of the FCR enzyme were not investigated.
Vacuum infiltration has been used in plant species where
removing the epidermis is difficult. However, incubation under
vacuum can cause modifications of the mesophyll tissue
metabolism. For instance, this procedure would remove to a
large extent CO2 and O2, therefore eliminating two major pathways for the dissipation of reducing power produced by the
photosynthetic electron transport chain. Therefore, vacuum
infiltration would likely cause an over-reduction of the mesophyll tissue. Methods less invasive, such as those involving the
removal of the epidermis, are therefore preferable when possible. When using peeled leaf disks, however, there are different
sources of Fe(III) reduction. The first is the FC-R enzyme(s)
present in the cell wall and plasma membrane of the mesophyll
cells. A second source is constituted by those compounds
released (leaked or excreted during incubation) from the mesophyll cells and the broken cells at the disk edge. A third source
is the pool of membranes and organelles in broken cells at the
disk edge, that are exposed to the medium and still capable of
performing enzymatic reactions normally carried out in the
cytoplasm.
The aim of this paper was to investigate the characteristics of the FC-R activities of mesophyll tissue from Fe-sufficient and -deficient sugar beet (Beta vulgaris L.). We used as
substrates Fe(III)-citrate and Fe(III)-malate, two of the most
likely carriers for Fe in the xylem, and the synthetic chelate
Fe(III)-EDTA. We measured the total Fe(III) reduction carried
out by leaf mesophyll disks devoid of the epidermis or incubated after vacuum infiltration. The effects of light and Fe deficiency on the mesophyll FC-R activity was measured at different pH values. The extent of the photochemical reduction of
Fe(III)-chelates was also measured. Finally, we have tried to
estimate separately several sources of Fe(III) reduction. The
kinetics of the Fe(III) reduction caused by mesophyll cells and
broken cells at the leaf disk margins has been also measured.
Materials and Methods
Plant material
Sugar beet (B. vulgaris L. hybrid Monohil, Hilleshög, Landskröna, Sweden) plants were grown in a growth chamber. Seeds were
germinated and grown in vermiculite for 2 weeks. Seedlings were
grown for two more weeks in half-strength Hoagland nutrient solution
with 45 mM Fe and then transplanted to 20 liter buckets (four plants
per bucket) containing half-strength Hoagland nutrient solution with 0
or 45 mM Fe. Iron was added as NaFe(III)-EDTA (Sigma, St. Louis,
MO, U.S.A.). In buckets with no Fe added the pH of the nutrient solu-
95
tion was raised with 1 mM NaOH and 1 g liter–1 of solid CaCO3 to
simulate conditions usually found in the field that lead to Fe deficiency; this treatment led to a constant pH of 7.7 throughout the 10–
12 d growth period (Susín et al. 1994). Plants were grown with a
PPFD of 350–400 mmol m–2 s–1 at 25°C, 80% relative humidity and a
photoperiod of 16 h light/8 h dark. Plants were used for measurements
1 week after transferring the plants to a Fe-free nutrient solution.
Young, rapidly expanding leaves were used for all measurements. All
chlorotic leaves sampled had no green veins, and showed a homogeneous color throughout the leaf.
Pigment analysis
Chl concentration per area was estimated non-destructively by
using a SPAD portable apparatus (Minolta Co., Osaka, Japan). To calibrate the SPAD, leaf disks were first measured with the SPAD, then
frozen in liquid N2, extracted with 100% acetone, and the extracts
were analyzed spectrophotometrically (Abadía and Abadía 1993).
Control leaves were chosen with SPAD values larger than 30
(300 mmol Chl m–2), whereas leaves from Fe-deficient plants were
chosen with SPAD values of approximately 8 (60 mmol Chl m–2).
Fe(III)-chelates
Fe(III)-EDTA was purchased from Sigma (St. Louis, MO,
U.S.A.). Fe(III)-citrate and Fe(III)-malate were prepared by dissolving
FeCl3 in citric acid-KOH or malic acid-KOH, pH 6.0, to give a final
stock of 5 mM Fe at the acid : Fe ratios desired (5 : 1 and 25 : 1 for citrate:Fe and malate:Fe, respectively). These organic anion : Fe ratios
lead to maximal FC-R activities with sugar beet leaf PM preparations
(González-Vallejo et al. 1999).
Ferric chelate reductase activity of leaf disks
The rates of Fe(III) reduction by leaf mesophyll disks were determined spectrophotometrically at 562 nm by measuring the formation
of the Fe(II)-PDTS complex from Fe(III)-EDTA, Fe(III)-citrate or
Fe(III)-malate, using an extinction coefficient of 27.9 mM–1 cm–1
(Cowart et al. 1993).
Preparation of leaf disks
Mesophyll disks were excised with a disk borer. Disks were
obtained from intact leaves with epidermis (Fig. 1A) and from leaf
pieces where the abaxial (lower) epidermis had been peeled off (Fig.
1B). The excision protocol produced a disk edge where broken cell
membranes were exposed to the medium (Fig. 1C). Removal of the
epidermis exposed more than one layer of mesophyll cells to the
medium (Fig. 1B). In a few cells (less than 1% of total cell number;
arrow in Fig. 1B) removal of the epidermis caused the rupture of the
cell wall and plasma membrane and internal organelles were also
exposed to the medium.
Two mesophyll disks (0.64 cm2 each) were incubated in 30 ml
glass vials with 10 ml of assay solution containing Fe(III)-chelate at
different concentrations and 400 mM PDTS. Measurements were made
in darkness or with red light illumination at different pH values. Light
was obtained from a halogen lamp located above the samples, and was
filtered with a red plastic filter (l >600 nm) and through 2 cm of icecold water. Light intensity at the sample level was 100 mmol m–2 s–1
PPFD, measured with a Skye (Powys, U.K.) PAR meter. Experiments
were generally run for 20 min under gentle shaking (Bühler SM orbital
shaker, speed 1.5). The ionic composition of the assay media mimicked that of the sugar beet apoplastic fluid (López-Millán et al. 2000),
and contained (in mM) 2.5 Mg2+, 6.5 Cl–, 1 Na+, 1 NH4+, 3.5 NO3–, 15
K+, 0.5 SO42– and 5 PO43–. Absorbance measurements were made at
20–25°C with a Shimadzu (Kyoto, Japan) model 2101-PC personal
computer-controlled double beam spectrophotometer using a 1 nm slit.
96
Leaf Fe(III) reduction and Fe deficiency
For obtaining the kinetics, the concentration of Fe(III)-chelate was varied in the range 62.5 to 2000 mM. In the experiment comparing three
reduction protocols (see below) the concentrations of chelates used
were 100 and 500 mM.
Estimates of the total reduction capacity, Tr, were made by using
disks obtained from leaf pieces where the abaxial epidermis had been
peeled off (method AI). A modification of method AI (method AII)
included a 30-min washing step with fresh assay medium. A vacuum
infiltration protocol (method B), similar to that used by de la Guardia
and Alcántara (1996), was carried out by using leaf disks with epidermis, first washed for 30 min with 0.5 mM CaSO4, 50 mM KH2PO4 and
1 mM KCl and then infiltrated with the same assay medium under vacuum (1700 Pa, Schött vacuum dessicator, 19 cm in diameter, 10 cm
high) with Fe(III)-chelate at the desired concentration and 400 mM
PDTS for 20 min. Vacuum was removed and re-applied 10 min after
the start of the incubation.
Blanks without leaf disks were always run under the same conditions with Fe(III)-chelates in the presence of PDTS, to estimate the
extent of photochemical reduction. For comparison with those of mesophyll, photochemical reduction rates were expressed on a leaf area
basis by using the volume/leaf area ratio used in the disk incubation
experiments. Mesophyll reduction rates presented in the paper are
always net, after subtracting the corresponding measured rates of photochemical reduction. Data were expressed on a leaf area basis, by
using only the area of the abaxial side. The FC-R activity found when
the assay solution containing Fe(III)-EDTA and PDTS was placed
directly for 20 min on the surface (adaxial or abaxial) of undamaged
leaves was negligible. Also, no measurable FC-R activity was found
with isolated leaf epidermis (not shown).
Sources of Fe(III) reduction
Not all of the total reduction by leaf disks originates from reduction at the mesophyll cells (Rem) exposed by the removal of the epidermis (Fig. 1B). A part of the total Fe(III) reduction activity is due to the
release to the incubation media of cell contents due to cell mechanical
damage at the edges of the mesophyll disks (Rlc; Fig. 1C). Also, cell
organelles, such as chloroplasts (Bughio et al. 1997, Mori 1998), mitochondria, etc., when exposed de novo, either at the disk edge (Fig. 1C)
or in some mesophyll cells open during epidermis removal (Fig. 1B),
may reduce Fe(III)-chelates enzymatically (Rec). Finally, a part of the
total Fe(III) reduction activity is associated with reducing compounds
released by mesophyll cells exposed to the media after the removal of
the epidermis (Rlm).
The procedures to measure or estimate the different sources of
Fe(III) reduction are described in Table 1. Total Fe(III) reduction
obtained from leaf disks where the abaxial epidermis had been peeled
off (Tr) was the sum of the four possible components Rem, Rec, Rlm and
Rlc. The total Fe(III) reduction of leaf disks with epidermis was taken
as the sum of the only two components associated to the leaf disk
edge, Rec and Rlc. The extent of reduction from released (excreted and/
or leaked) compounds was measured by incubating leaf disks in the
assay solution lacking Fe(III)-chelates and PDTS, removing the disks,
adding Fe(III)-chelates and PDTS to the assay medium and finally
measuring the Fe reducing capacity of the medium, which contained
the reducing compounds released from the mesophyll tissue. These
rates of reduction will correspond to Rlc when disks with epidermis
were used and the sum of Rlc and Rlm for disks without epidermis
(Table 1). The true enzymatic activity from intact mesophyll cells was
then calculated as Rem = Tr – Rec – Rlm – Rlc. Assays were done in all
cases at the average pH found in sugar beet apoplast (pH 6.0; LópezMillán et al. 2000) and at Fe(III)-chelate concentrations from 62.5 to
2000 mM.
Fig. 1 Scanning electron micrograph showing the leaf epidermal surface (A), the exposed mesophyll cells after removal of the epidermis
(B) and the disc excision edge in a leaf disk with epidermis (C). The
arrow in B indicates a mesophyll cell broken during the epidermis
removal step. The arrow in C indicates the cut disk edge. All micrographs are from abaxial surfaces of control sugar beet leaves.
Leaf Fe(III) reduction and Fe deficiency
Table 1
97
Sources of Fe(III) reduction
Terminology
Measured/
estimated
Description
Assay
Calculation
Tr: Rec+Rlc+Rem+Rlm measured
total reduction capacity
mixture of components
–
Rec+Rlc
Rlc+Rlm
measured
measured
mixture of components
mixture of components
Rlc
measured
non-enzymatic reduction due to
substances excreted by or leaked
from broken and intact cells at
the excision edge
Rlm
estimated
Rec
estimated
Rem
estimated
non-enzymatic reduction due to
substances excreted by or leaked
from mesophyll cells
enzymatic reduction by intact
cells and cell membranes and
organelles from broken cells
exposed at the excision edge
true enzymatic reduction by the
plasma membranes of mesophyll cells
leaf disks where the abaxial
(lower) epidermis had been
peeled off
leaf disks with epidermis
incubating leaf disks without epidermis in assay solution lacking
Fe(III)-chelates and PDTS,
removing the disks, adding
Fe(III)-chelates and PDTS to the
assay medium
incubating leaf disks with epidermis in the assay solution lacking
Fe(III)-chelates and PDTS,
removing the disks, adding
Fe(III) chelates and PDTS to the
assay medium
–
Organic anion analyses
With the volume to leaf area ratio used in the reductase experiments the amount of organic acids in solution was too low to permit
adequate quantification. Therefore, we incubated eight mesophyll
disks (0.64 cm2 each) in 1 ml of basic assay medium for 60 min, disks
were then removed and the assay solution was filtered through a
0.2 mM Millipore filter. Samples were analyzed immediately by HPLC
with a 300´7.8 mm Aminex ion-exchange column (HPX-87H, BioRad Laboratories, Hercules, CA, U.S.A.) with a HPLC Waters system, including a 600E pump, a 996 photodiode array detector and the
Millennium 2010 software. Samples were injected with a Rheodyne
injector (20 ml loop). Mobile phase (8 mM sulfuric acid) was pumped
with a 0.6 ml min–1 flow rate. Organic anions were detected at 210 nm.
Peaks corresponding to oxalate, cis-aconitate, citrate, 2-oxoglutarate,
malate and fumarate were identified by comparison of their retention
times with those of known standards from Bio-Rad Laboratories (Hercules, CA, U.S.A.) and Sigma (Saint Louis, MO, U.S.A.). Spectral
analysis of the peaks was used to confirm their identification. Quantification of the released amounts of each organic anion per leaf area was
made with known amounts of each compound using peak areas.
Electron microscopy
Electron microscopy was performed on leaf disk sections fixed in
2.5% glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.4) for 2 h at
room temperature, 20 h at 4°C and then washed three times in the
–
–
–
(Rlc+Rlm) – Rlc
–
(Rec+Rlc) – Rlc
–
Tr – (Rec + Rlm + Rlc)
same buffer and twice in ultrapure water. After dehydration in acetone
series, samples were critical-point dried, gold-palladium coated and
viewed at 10 kV in a LEO 430 (LEO Electron Microscopy Ltd., Cambridge, U.K.) scanning electron microscope.
Results
Iron reduction by mesophyll disks with three different protocols
Three protocols were used to measure the FC-R activity of
illuminated leaf mesophyll disks. Measurements were carried
out with unwashed, peeled disks (protocol AI), washed, peeled
disks (protocol AII) and vacuum-infiltrated, unpeeled disks
(protocol B). The highest FC-R rates were obtained with AI,
followed by AII and then B (Fig. 2). This occurred with both
Fe-sufficient and Fe-deficient leaf disks using 100 mM (data
not shown) and 500 mM Fe(III)-EDTA (Fig. 2A), Fe(III)-citrate (Fig. 2B) and Fe(III)-malate (Fig. 2C). Controls with
unpeeled disks treated with a protocol otherwise similar to AI
were also carried out and gave much lower reduction values
(see Table 3 below). Also, controls with peeled disks infiltrated under vacuum were carried out and gave similar results
98
Leaf Fe(III) reduction and Fe deficiency
Table 2 Iron(III)-chelate reduction activity in Fe-sufficient
(+Fe) and Fe-deficient (–Fe) sugar beet mesophyll disks on a
Chl basis (nmol Fe reduced (nmol Chl)–1 min–1) using Fe(III)EDTA, Fe(III)-citrate and Fe(III)-malate as Fe sources
Fe(III)-EDTA
+Fe
–Fe
–Fe/+Fe
Fe(III)-citrate
+Fe
–Fe
–Fe/+Fe
Fe(III)-malate
+Fe
–Fe
–Fe/+Fe
Light
Dark
0.018±0.002
0.048±0.005
2.7
0.003±0.001 (17%)
0.011±0.001 (23%)
3.6
0.026±0.007
0.042±0.006
1.6
0.004±0.001 (15%)
0.019±0.001 (45%)
4.8
0.028±0.002
0.094±0.012
3.4
0.003±0.001 (11%)
0.014±0.001 (15%)
4.7
Measurements were made at optimum pH either in darkness or
in illuminated mesophyll disks (100 mmol photons m–2 s–1 of red
light), with 500 mM Fe(III)-chelate as Fe source. Leaf Chl concentrations were 362±17 and 58±3 mmol m–2 in the Fe-sufficient
and Fe-deficient disks, respectively. Data are mean±SE of nine
replications. In parenthesis the percentage of total reduction
occurring in the dark.
Fig. 2 Iron reduction activity of Fe-sufficient (+Fe) and Fe-deficient
(–Fe) sugar beet leaf mesophyll disks using Fe(III)-EDTA (A), Fe(III)citrate (B) or Fe(III)-malate (C) as Fe sources. Measurements were
made with three different methods (AI, AII and B; see text for further
explanation), with 500 mM Fe(III)-chelate at pH 6.0. Leaf disks were
illuminated by 100 mmol m–2 s–1 PPFD red light. Data are mean±SE of
six replications.
than those found with protocol AI (not shown). For further
measurements method AI was used.
Characteristics of the reduction of Fe(III)-chelates by
mesophyll disks
Reduction of Fe(III)-EDTA, Fe(III)-citrate and Fe(III)malate by mesophyll disks was markedly enhanced by light
(Fig. 3A–C). At the optimal pH values, light increased the FCR rates of Fe-deficient mesophyll disks 5-, 2- and 4-fold with
Fe(III)-EDTA, Fe(III)-citrate and Fe(III)-malate, respectively.
With control disks light-induced increases were 6-fold with
Fe(III)-EDTA and 9-fold with Fe(III)-citrate and Fe(III)-malate
(Fig. 3A–C).
In the light the FC-R rates of Fe-sufficient mesophyll
disks depended on the pH of the assay medium (Fig. 3A–C),
whereas in Fe-deficient disks the optimal pH were less marked.
The pH optima found for control disks were different for the
three chelates tested. Maximal reduction rates for Fe(III)EDTA (0.57 nmol Fe reduced cm–2 min–1) were found at
pH 6.0 (Fig. 3A). For Fe(III)-citrate the pH optimum was 6.2
(Fig. 3B), with a maximal FC-R activity of 0.90 nmol Fe
reduced cm–2 min–1, and for Fe(III)-malate the optimum was
pH 6.7 (Fig. 3C), with maximal reduction rates of 0.87 nmol Fe
reduced cm–2 min–1. When Fe reduction was carried out in
darkness, pH dependence was not observed in control or Fedeficient mesophyll disks (Fig. 3A–C).
Iron deficiency decreased markedly the FC-R rates in the
light when expressed on a leaf area basis (Figs. 2, 3A–C).
Decreases in the FC-R rates with Fe deficiency were approximately 55% for Fe(III)-malate and Fe(III)-EDTA and 80% in
the case of Fe(III)-citrate. Conversely, Fe deficiency had only
small effects on the dark FC-R rates, which were similar to
those found with control mesophyll disks (Fig. 3A–C).
When expressed on a Chl basis, however, Fe deficiency
increased the FC-R rates of illuminated mesophyll disks (Table
2). Rates increased approximately 2.7-fold with Fe(III)-EDTA,
Leaf Fe(III) reduction and Fe deficiency
99
Fig. 3 pH dependence of the Fe(III)-chelate reduction activities. (A–C) sugar beet mesophyll Fe-sufficient (squares) and Fe-deficient (circles)
disks, using Fe(III)-EDTA (A), Fe(III)-citrate (B) or Fe(III)-malate (C). (D–F) photochemical reduction (triangles) in the absence of leaf disks
with Fe(III)-EDTA (D), Fe(III)-citrate (E) or Fe(III)-malate (F). Measurements were made in darkness (solid symbols) or with 100 mmol photons
m–2 s–1 of red light (open symbols), with 500 mM Fe(III)-chelate. Data are mean±SE of nine replications.
1.6-fold with Fe(III)-citrate and 3.4-fold with Fe(III)-malate.
The small increase found with Fe(III)-citrate on a Chl basis
reflects the major decrease caused by Fe deficiency in the rate
of reduction of this chelate per area basis. In darkness, Fe-deficient mesophyll disks also reduced more Fe than the controls
when data were expressed on a Chl basis (4- to 5-fold, Table
2). The apparent increases in reduction rates with Fe-deficient
disks on a Chl basis are likely to be due to their very low Chl
content (leaf disks had 360 and 60 mmol Chl m–2 in the Fesufficient and Fe-deficient disks, respectively; Table 2).
Photochemical reduction of Fe(III)-chelates
Photochemical reduction of Fe(III)-EDTA was approximately 10% of the total mesophyll reduction (Fig. 3D). When
expressed on a leaf surface basis, photochemical reduction in
the absence of leaf material was equivalent to a maximum of
0.05 nmol Fe reduced cm–2 min–1 at pH values lower than 6.5
(Fig. 3D). Photochemical reduction of Fe(III)-malate was equivalent to approximately 17% of the FC-R rates obtained with
mesophyll disks. Rates were maximal at pH values below 7,
with values of approximately 0.15 nmol Fe reduced cm–2 min–1
(Fig. 3F).
100
Leaf Fe(III) reduction and Fe deficiency
Table 3 Sources of Fe(III) reduction in Fe-sufficient (+Fe) and Fe-deficient (–Fe) sugar beet
leaves
Tr
Rec + Rlc
Rlm + Rlc
Rlc
Rlm
Rem
Rec
0.101
0.107
0.167
0.287
0.404
0.423
0.066
0.051
0.061
0.152
0.102
0.107
0.017
0.014
0.021
0.037
0.015
0.020
0.049
0.037
0.039
0.115
0.087
0.087
0.027
0.034
0.046
0.131
0.459
0.497
0.084
0.093
0.147
0.250
0.389
0.403
0.058
0.069
0.088
0.138
0.201
0.229
0.041
0.044
0.061
0.056
0.065
0.061
0.007
0.008
0.019
0.024
0.026
0.027
0.034
0.036
0.042
0.032
0.039
0.034
–
–
0.055
0.105
0.112
0.094
0.051
0.061
0.069
0.114
0.176
0.202
0.120
0.132
0.134
0.178
0.160
0.229
0.061
0.056
0.114
0.116
0.175
0.064
0.037
0.037
0.050
0.077
0.093
0.050
0.024
0.019
0.064
0.039
0.082
0.014
0.158
0.189
0.161
0.430
0.481
0.620
0.083
0.095
0.084
0.101
0.067
0.179
0.073
0.081
0.085
0.091
0.124
0.162
0.049
0.049
0.071
0.111
0.129
0.070
0.037
0.037
0.050
0.078
0.093
0.050
0.012
0.012
0.021
0.033
0.036
0.020
0.023
0.058
0.099
0.108
0.195
0.204
0.036
0.044
0.035
0.013
0.031
0.112
0.121
0.161
0.217
0.314
0.478
0.622
0.047
0.079
0.067
0.130
0.166
0.140
0.040
0.055
0.040
0.105
0.114
0.102
0.007
0.024
0.027
0.025
0.052
0.038
0.049
0.147
0.228
0.264
0.286
0.541
0.081
0.106
0.177
0.209
0.364
0.520
0.026
0.029
0.077
0.121
0.145
0.226
0.090
0.087
0.050
0.100
0.059
0.023
0.049
0.056
0.026
0.028
0.012
0.049
0.041
0.031
0.024
0.072
0.047
–
0.012
0.028
0.034
0.119
0.153
0.155
–
–
0.051
0.093
0.133
0.177
Fe(III)-EDTA (mM)
62.5
125
250
500
1000
2000
62.5
125
250
500
1000
2000
+Fe
0.177
0.178
0.253
0.533
0.950
1.007
–Fe
0.068
0.058
0.185
0.275
0.353
0.357
Fe(III)-citrate (mM)
62.5
125
250
500
1000
2000
62.5
125
250
500
1000
2000
+Fe
0.302
0.340
0.359
0.647
0.723
0.863
–Fe
0.108
0.151
0.205
0.232
0.355
0.386
Fe(III)-malate (mM)
62.5
125
250
500
1000
2000
62.5
125
250
500
1000
2000
+Fe
0.177
0.332
0.472
0.603
0.816
1.201
–Fe
0.102
0.115
0.135
0.312
0.345
0.381
Total reduction (Tr) was considered to be the sum of four components, Rem (mesophyll enzymatic), Rec (disk edge enzymatic), Rlm (mesophyll leakage) and Rlc (disk edge leakage). Measurements were made with 100 mmol photons m–2 s–1 of red light at pH 6.0. Values are in nmol Fe
reduced cm–2 min–1. Data are means of nine measurements.
Leaf Fe(III) reduction and Fe deficiency
Fig. 4 Relative contribution of the different sources of Fe reduction
in Fe-sufficient (+Fe) and Fe-deficient (–Fe) sugar beet mesophyll
disks, using 500 mM Fe(III)-EDTA, Fe(III)-citrate or Fe(III)-malate as
Fe sources. Measurements were made in illuminated mesophyll disks
(100 mmol photons m–2 s–1 of red light). Areas are proportional to the
Fe(III)-chelate reduction activity.
Photochemical reduction of Fe(III)-citrate in the absence
of leaf disks, however, was much higher than those of Fe(III)EDTA and Fe(III)-malate, and was in the same order of magnitude as the reduction due to leaf mesophyll disks (Fig. 3E). The
rate of reduction was dependent on pH, with a maximum of
approximately 1 nmol Fe reduced cm–2 min–1 at pH 6.2.
Sources of reduction of Fe(III)-chelates
Reduction of Fe(III)-chelates by compounds released from
the leaf edge (Rlc) did not show enzymatic kinetics. With
500 mM Fe(III)-EDTA, Rlc accounted for approximately 7 and
9% of the total Fe(III) reduction by illuminated Fe-sufficient
and Fe-deficient mesophyll disks, respectively (Table 3). Rlc
accounted in Fe-sufficient and Fe-deficient mesophyll disks for
approximately 12 and 34% of the total reduction with Fe(III)citrate and for 17 and 9% with Fe(III)-malate (Table 3).
101
Reduction of Fe(III)-chelates by compounds released from
the leaf mesophyll surface (Rlm) was also non-enzymatic. With
500 mM Fe(III)-EDTA Rlm accounted for approximately 22 and
12% of the total Fe(III) reduction by illuminated Fe-sufficient
and Fe-deficient mesophyll disks, respectively (Table 3). Rlm
accounted in Fe-sufficient and Fe-deficient mesophyll for
approximately 6 and 14% of the total reduction with Fe(III)citrate and for 4 and 23% with Fe(III)-malate.
The Fe(III) reduction activity associated to the disk edges
(Rec) had enzymatic kinetics with the three chelates tested. This
accounted for approximately 47 and 41% of the reducing activity of Fe-sufficient and deficient leaf disks, respectively, with
500 mM Fe(III)-EDTA (Table 3). With Fe(III)-citrate Rec
accounted for approximately 16 and 6% of the Fe reducing
activity in Fe-sufficient and deficient plants, respectively. With
Fe(III)-malate Rec accounted for approximately 35 and 30% of
the Fe reduction activity in Fe-sufficient and deficient plants,
respectively.
The true reduction by mesophyll cells (Rem) had Michaelis-Menten kinetics with the three Fe(III)-chelates tested. With
Fe(III)-EDTA Rem accounted for approximately 24 and 38% of
the total reduction by illuminated Fe-sufficient and Fe-deficient mesophyll disks, respectively (Table 3). With Fe(III)-citrate and Fe(III)-malate Rem accounted for approximately 66–
47% and 44–38% of the total reduction in Fe-sufficient and Fedeficient disks, respectively.
Therefore, in illuminated control and Fe-deficient mesophyll disks at pH 6.0 and 500 mM Fe(III)-EDTA the largest Fe
reduction source was the enzymatic reduction by the leaf disk
cut edge (Rec), followed by the enzymatic reduction of the mesophyll (Rem) and the reduction due to compounds released by
the mesophyll (Rlm) and the disk edge (Rlc) (Fig. 4). Under the
same conditions but with Fe(III)-citrate Rem was always the
largest component. In control disks this was followed by Rec,
Rlc and then Rlm, and in Fe-deficient disks the order was
Rem>Rlc>Rlm>Rec (Fig. 4). With Fe(III)-malate the relative
importance was Rem>Rec>Rlc>Rlm for control disks and
Rem>Rec>Rlm>Rlc for Fe-deficient disks (Fig. 4).
Reduction of Fe(III)-EDTA by oxalate
Organic anions released by Fe-deficient mesophyll disks
were mainly oxalate, citrate and malate, with minor amounts of
cis-aconitate, 2-oxoglutarate and fumarate (Table 4). When
using Fe-sufficient mesophyll disks organic acids found were
mainly oxalate and malate, with small amounts of citrate and 2oxoglutarate (Table 4).
Solutions containing only oxalate, the major organic anion
released from Fe-sufficient and Fe-deficient mesophyll disks,
were able to reduce Fe(III)-EDTA in the light (Table 5). Pure
oxalate, at concentrations similar to those released during incubation by mesophyll disks, was able to reduce approximately
0.18 and 0.06 nmol Fe cm–2 min–1, values similar to those
found with media where leaf disks had been incubated.
102
Leaf Fe(III) reduction and Fe deficiency
Table 4 Amounts of organic acids (in mmol cm–2 leaf area)
excreted by Fe-sufficient (+Fe) and Fe-deficient (–Fe) sugar
beet mesophyll disks (mean±SE of four measurements)
Oxalate
cis-Aconitate
Citrate
2-Oxoglutarate
Malate
Fumarate
+Fe
–Fe
0.45±0.04 (288.0)
ND a
0.006±0.001 (4.1)
0.003±0.001 (1.6)
0.067±0.001 (42.8)
ND
0.25±0.01 (161.0)
0.008±0.001 (5.3)
0.099±0.010 (63.8)
0.003±0.001 (2.1)
0.082±0.003 (52.3)
0.001±0.001 (0.8)
Pure oxalate solution a
Assay medium after disk incubation
288 mM
161 mM
+Fe
–Fe
0.18±0.01 0.06±0.01 0.15±0.01
0.06 ±0.01
These assay media contained compounds released from the broken cells at the cut edge and from undamaged mesophyll cells
exposed to the medium after the removal of the epidermis. Data
are mean±SE, of 6–9 replications and are expressed in nmol Fe
reduced cm–2 min–1. The pH of the assay medium was 6.4.
In parenthesis the corresponding organic anion concentrations
(in mM) in the assay medium for the volume/leaf area ratio used
in the reductase experiments.
a
Table 5 Iron reduction from 500 mM Fe(III)-EDTA by solutions of pure oxalate and by assay media after leaf disk incubation and disk removal
a
The raw values of Fe reduction by oxalate were transformed into values on a leaf surface basis by using the volume to leaf area ratio used in
all experiments.
ND = not detected.
Kinetic characteristics of the Fe(III)-chelate reductase
Michaelis-Menten kinetics were found for Tr, Rem and Rec
for Fe-deficient and control disks and all three chelates tested
(Table 6). The Km values for total mesophyll reduction (Tr) did
not show significant differences for Fe(III)-EDTA and Fe(III)citrate between the treatments, whereas for Fe(III)-malate a
higher affinity was found for Fe-deficient mesophyll disks. The
Vmax for the total reduction decreased with Fe deficiency (Table
6).
For the estimated true mesophyll enzymatic reduction
(Rem) Km values showed a different picture: with Fe(III)-EDTA
affinity was higher in Fe-deficient mesophyll disks, in contrast
to Fe(III)-citrate and Fe(III)-malate which showed higher affinities in Fe-sufficient mesophyll disks. The Rem Vmax decreased
with Fe deficiency (Table 6).
For the estimated enzymatic activity at the disk edge (Rec),
the highest affinities were found for Fe(III)-citrate. With
Fe(III)-EDTA affinity was higher under Fe deficiency, whereas
with Fe(III)-malate a lower Km value was found under sufficient Fe supply. The Rec Vmax decreased with Fe deficiency
(Table 6). It should be mentioned that when using Fe(III)-citrate constants should be taken with care, since r values
obtained are very low. This is possibly due to sucessive errors
introduced in calculation of Rec. In the case of citrate errors
may be larger because raw values had to be corrected for very
high photochemical reduction values.
Discussion
The rates of Fe(III) reduction by sugar beet illuminated
Table 6 Enzymatic characteristics of the total reduction, the true mesophyll reduction (Rem) and
the disk edge reduction (Rec) estimated from data shown in Table 3
Km
Fe(III)-EDTA
Vmax
r
Km
Total reduction
+Fe
317
0.927
0.67
114
–Fe
297
0.383
0.71
174
–Fe/+Fe 0.94
0.41
1.52
True mesophyll enzymatic reduction (Rem)
+Fe
545
0.300
0.26
164
–Fe
136
0.111
0.44
437
–Fe/+Fe 0.25
0.37
2.66
Disk edge enzymatic reduction (Rec)
+Fe
294
0.412
0.82
25
–Fe
189
0.178
0.78
19
–Fe/+Fe 0.64
0.43
0.76
Fe(III)-citrate
Vmax
r
Km
Fe(III)-malate
Vmax
r
0.757
0.378
0.50
0.79
0.92
358
190
0.53
1.207
0.366
0.30
0.93
0.77
0.513
0.241
0.47
0.70
0.84
327
459
1.40
0.468
0.162
0.35
0.71
0.62
0.113
0.049
0.43
0.32
0.12
362
1012
2.80
0.478
0.268
0.56
0.83
0.98
Constants were calculated with the Eadie-Hofstee method. Km and Vmax values are in mM and nmol
Fe reduced cm–2 min–1, respectively. Data are the mean of 6–9 measurements.
Leaf Fe(III) reduction and Fe deficiency
leaf disks were dependent on the methodology used. We compared our method using peeled leaf disks with a method using
vacuum infiltration that has been used before for the same purposes (Brüggemann et al. 1993, de la Guardia and Alcántara
1996). Method AI (unwashed mesophyll disks without epidermis) gave the highest Fe reduction rates, followed by method
AII (washed mesophyll disks without epidermis) and then
method B (vacuum-infiltrated leaf disks with epidermis). Methods AI and AII differ only in a brief washing step and, therefore,
differences found must arise from the removal of reducing
compounds released from the broken cells at the disk edge. The
fact that rinsing removes more Fe reduction activity with
Fe(III)-citrate and Fe(III)-malate than with Fe(III)-EDTA suggests that the reducing compounds released from the broken
cells at the disk edge reduce better natural chelates than the
synthetic Fe-chelates. A possible explanation could be the
existence of a soluble ferric citrate reductase (Sparla et al.
1999). The low reducing rates found with method B (vacuuminfiltrated leaf disks with epidermis) indicate that removal of
the epidermis is crucial to measure high mesophyll reduction
rates in plant species where this procedure is feasible.
The sugar beet mesophyll Fe(III) reduction activity was
markedly light-dependent. The increase in FC-R activity with
light was dependent on the Fe(III)-chelate used, and was in the
ranges 2- to 5-fold and 6- to 9-fold with Fe-deficient and control mesophyll disks, respectively. These increases were similar to those found before in leaf pieces of Vigna unguiculata (3fold; Brüggemann et al. 1993) and sunflower (10-fold; de la
Guardia and Alcántara 1996), but smaller than those found
with isolated sugar beet protoplasts (35-fold; González-Vallejo
et al. 2000).
Iron-deficient and Fe-sufficient sugar beet mesophyll
disks were able to reduce Fe(III)-malate and Fe(III)-citrate.
Fe(III)-malate has been recently reported to be reduced by
kiwifruit leaf disks (Rombolà et al. 2000) and isolated PM
from sugar beet leaves (González-Vallejo et al. 1999). Higher
FC-R rates with Fe(III)-citrate than with Fe(III)-EDTA were
also found in mesophyll disks of V. unguiculata (Brüggemann
et al. 1993). The finding that Fe(III) malate can be efficiently
reduced by mesophyll tissue could be crucial to understand the
physiology of Fe-deficient plants. In sugar beet, the concentration of citrate and malate in the xylem sap increase 26- and
14-fold, respectively, by comparison to the controls (LópezMillán et al. 2000).
Photochemical reduction of Fe(III) occurred with the three
Fe(III)-chelates tested. In the case of Fe(III)-malate and Fe(III)EDTA photochemical reduction rates accounted for less than
10% of the total reduction. However, the photochemical reduction rates for Fe(III)-citrate were similar or even higher than
the mesophyll reduction rates. This supports the hypothesis that
photochemical reduction of Fe(III)-citrate in stems and leaves
may occur during Fe transport to the leaves, as it was suggested
by Bienfait and Scheffers (1992). This process would be potentially more important in those plant organs that are more
103
exposed to a high PPFD, such as the leaf lamina. Furthermore,
this process would be more likely in leaves with low Chl concentration. In low-Chl, Fe-deficient leaves of sugar beet, pear
and peach even the lower layers of cells in the leaf are exposed
to a relatively high PPFD, that may be as high as 20–40% of
the incident PPFD (Morales et al. 1991, Abadía et al. 2000).
The optimal pH for the sugar beet mesophyll FC-R activity was in the range 6.0–6.7, a pH commonly found in the apoplastic space of sugar beet leaves (López-Millán et al. 2000).
These values are lower than those found previously for optimal FC-R activity in leaf PM isolated from sugar beet
(González-Vallejo et al. 1999) and V. unguiculata (Brüggemann
et al. 1993). Optimum pHs for the leaf mesophyll FC-R activity
with Fe(III)-citrate were 7.6–6.8 in Valerianella locusta, 6.9–
6.7 in Prunus persica and 6.2–5.8 in B. vulgaris (Grünewald
1996). It has been suggested that the FC-R enzyme could have
a strict pH regulation (Mengel 1995), and that the increase in
apoplastic pH induced by Fe deficiency would lead to the inactivation of the enzyme, preventing Fe uptake by the mesophyll
cells and producing the accumulation of Fe in the apoplast
(Kosegarten et al. 1999). Our data indicate, however, that
decreases in Fe(III) reduction activity would occur only at very
high pH values, which have not been detected so far in the
apoplast of Fe-deficient leaves (López-Millán et al. 2000).
Our results support that Fe deficiency causes a marked
decrease of the FC-R activity in sugar beet on a leaf area basis.
This is in good agreement with the decrease in FC-R activity
on a protoplast surface basis found in isolated sugar beet protoplasts (González-Vallejo et al. 2000). Previous data indicated
that Fe deficiency did not increase leaf mesophyll FC-R activity on an area basis in V. unguiculata (Brüggemann et al. 1993)
and decreased leaf FC-R activities by 40% on a fresh mass
basis in sunflower (de la Guardia and Alcántara 1996) and in
vacuum-infiltrated leaf disks of kiwi (Rombolà et al. 2000).
Therefore, there is no evidence so far of any Fe deficiencymediated induction of the FC-R activity in leaves, conversely
to the strong induction of that enzyme in the roots of the same
plants (Schmidt 1999).
Ferric chelate reductase activities have usually been
expressed on a Chl basis because of the light dependence of the
reductase. Activities on a Chl basis found in the present work
increased with Fe deficiency approximately 3-fold with Fe(III)EDTA and Fe(III)-malate and 2-fold with Fe(III)-citrate. When
activities are expressed on a Chl basis, Fe deficiency has been
reported to cause 3-, 6-, 16- and 4-fold increases in the mesophyll FC-R activity of V. unguiculata, Valerianella locusta,
Prunus persica and B. vulgaris leaf pieces (Brüggemann et al.
1993, Grünewald 1996), and only small (5%) increases in H.
annuus leaf disks (de la Guardia and Alcántara 1996).
The FC-R activities of leaf disks were lower than those
obtained with protoplasts isolated from similar leaves
(González-Vallejo et al. 2000). Under illumination, using
Fe(III)-EDTA as Fe source and at optimum pH, the Vmax of
control and Fe-deficient sugar beet protoplasts were 0.11 and
104
Leaf Fe(III) reduction and Fe deficiency
0.17 nmol Fe reduced nmol–1 Chl min–1, respectively (calculated from González-Vallejo et al. 2000), whereas under the
same conditions the Vmax for the total reduction of control and
Fe-deficient sugar beet were 0.03 and 0.07 nmol Fe reduced
nmol–1 Chl min–1, respectively (calculated from data in Table
6). This indicates that the maximum reducing capacity (i.e. that
obtained with protoplasts) is never attained with leaves, possibly because of limited accessibility of the solution to the whole
surface of the plasma membrane.
The Km reported in this work for the mesophyll FC-R
(114–545 mM) are within the same order of magnitude of those
found in previous works. In V. unguiculata, the mesophyll FCR activity had a higher apparent specificity for the natural substrate Fe(III)-citrate (Km 49–56 mM) than for the synthetic
chelate Fe(III)-EDTA (Km 226 mM) (Brüggemann et al. 1993).
In the same work, it was reported that Fe deficiency did not
change the Km for Fe(III)-citrate. Grünewald (1996) gave Km
values of approximately 22 and 48 mM for Fe(III)-citrate in Fesufficient and deficient leaves of Valerianella locusta.
Our data show that the measurement of FC reduction by
leaf mesophyll disks included not only the true enzymatic
reduction at the surface of the mesophyll cells, but also reduction arising from several other sources. In fact, true enzymatic
mesophyll reduction was only 24–66% and 38–47% of the total
rates in Fe-sufficient and Fe-deficient leaf disks, respectively.
Mesophyll cells are also able to reduce Fe-chelates non-enzymatically by the release of reducing components, which can
account for 6–23% of the total reduction, depending on the
source of Fe. This was shown by the lack of Michaelis-Menten
kinetics, and was probably related to compounds released to
the solution such as oxalate (Table 5). The presence of compounds capable to reduce Fe(III) in the medium where leaf
pieces had been incubated has been reported previously
(Abadía et al. 1984, Mehrotra and Gupta 1990).
As leaf disks always contain edges, two other sources,
immanent to the method, were found, which cannot be avoided
when leaf disks are used. Enzymatic reduction at the edge of
the disks (Fig. 1C), which had a surface equivalent to only
approximately 16% of the abaxial surface, accounted for 30–
47% of the total reduction with Fe(III)-EDTA and Fe(III)malate and for 6–16% with Fe(III)-citrate. This activity indicates that there is a strong enzymatic activity at the leaf disk
margins with some Fe sources, likely due to organelles exposed
to the medium at the disk cut edge (Fig. 1C). Possibly this
could be caused by chloroplasts (Bughio et al. 1997, Mori
1998) and other organelles. Although this does not occur with
Fe(III)-citrate, the apparent affinity of the FC-R associated to
the leaf edges with this substrate was much higher than that
found to be associated with the true mesophyll activity. Furthermore, disk edges also showed a release of reducing compounds accounting for 7–34% of the total reduction.
The multi-component origin of the Fe(III)-chelate reduction by leaf mesophyll disks is a serious constraint that strongly
suggests leaf disks should only be used when every different
reduction source is estimated. In our case, however, the effect
of Fe deficiency on the Vmax was similar for the total reduction
and the true mesophyll enzymatic reduction. The estimation of
the kinetic characteristics could be also affected by the multicomponent origin. For instance, low Km values for the disk
edge enzymatic reduction would contribute to decrease the Km
estimated for the total reduction.
Acknowledgement
Supported by grants AGR97–1177 from the Comisión Interministerial de Ciencia y Tecnología to A.A., and PB94–0086 from the
Dirección General de Investigación Científica y Técnica and AIR3CT94–1973 from the Commission of European Communities to J.A.
A.L. was supported by fellowships from the International Center for
Advanced Mediterranean Studies-Agronomic Mediterranean Institute
of Zaragoza and the Spanish Institute of International Cooperation. A.F.L.-M. was supported by a fellowship from the Spanish Ministry of
Science and Education. F.M. and Y.G. were scientists on contracts
from the Spanish Ministry of Education and Culture and the Spanish
Council for Scientific Research, respectively. Authors gratefully
acknowledge the skilful technical assistance of Aurora Poc and Pilar
Zanuy, and thank Dr. Juan Marin for use of equipment.
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(Received March 13, 2000; Accepted November 8, 2000)