Plant Physiology and Biochemistry 41 (2003) 27–33
www.elsevier.com/locate/plaphy
Original article
Flux of protons released by wild type and ferritin over-expressor
tobacco plants: effect of phosphorus and iron nutrition
G. Vansuyt a, G. Souche b, A. Straczek b, J.-F. Briat a, B. Jaillard b,*
a
UMR 5004 CNRS, université Montpellier II, ENSA-M, INRA biochimie et physiologie moléculaire des plantes,
place Pierre-Viala, 34060 Montpellier cedex 1, France
b
UMR 388 ENSA-M, INRA sol et environnement, place Pierre-Viala, 34060 Montpellier cedex 1, France
Received 10 June 2002; accepted 31 July 2002
Abstract
Tobacco (Nicotiana tabacum) plants over-expressing the iron storage protein ferritin, either in the cytoplasm or in the plastids, were grown
under various P and Fe conditions. The crossed effects of both the genotypes and the environmental conditions on iron and chlorophyll
concentrations in leaves, ferric reductase (EC 1.6.99.13) and plasmalemma H+-ATPase (EC 3.6.3.6) activities in roots, and fluxes of H+
released by roots were determined. The increase in leaf Fe concentration observed in plants over-expressing ferritin was accompanied by an
increase in root ferric reductase and H+-ATPase activities. Iron deficient conditions induced a decrease in Fe and chlorophyll concentrations
in leaves, an increase in ferric reductase and H+-ATPase activities in roots and an increase in H+-flux released by roots in all genotypes.
Phosphorus abundant conditions induced also an increase in ferric reductase and H+-ATPase activities in roots in all genotypes, and an increase
in H+-flux released by roots in the genotype over-expressing ferritin in the cytoplasm. These results suggest that P could regulate the root Fe
uptake system, at the gene expression level, as already reported for Zn in barley. Moreover, they show that H+-flux was not always consistent
with H+-ATPase activity, revealing that the global measurement of root plasmalemma H+-ATPase activity has some limits compared to the
localized determination of the H+-flux. These limits could be due to the fact that part of the H+-flux is not directly linked to H+-ATPase activity
in roots.
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
Keywords: Ferritin over-expressor; H+-ATPase activity; Iron deficiency; Phosphorus nutrition; Proton release; Reductase activity; Tobacco
1. Introduction
Non-graminaceous plants react to iron deficiency by activating a complex process evolved for mining this metal from
the soil [12]. This process is located along the root, within the
subapical zone where iron is taken up by the plant [13,16].
First rhizosphere is acidified through activation of a specific
root H+-ATPase (EC 3.6.3.6), likely to be encoded by the
aha2 gene in Arabidopsis thaliana (M.L. Guerinot, personal
Abbreviations: CaMV, cauliflower mosaic virus; Cd, cadmium; DW, dry
weight; Fe, iron; Fe-EDTA, ethylene-diamine-tetra-acetic acid ironIII sodium salt; FW, fresh weight; GUS, beta-D-glucuronidase (EC 3.2.1.31);
H+-ATPase, proton-translocating P-type ATPase (EC 3.6.3.6); IRT, iron
regulated transporter; Nramp, natural resistance associated macrophage
protein; P, phosphorus; Zn, zinc.
* Corresponding author.
E-mail address: [email protected] (B. Jaillard)
© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.
PII: S 0 9 8 1 - 9 4 2 8 ( 0 2 ) 0 0 0 0 5 - 0
communication). The local pH decrease induces an increase
in solubility of FeIII-hydroxides and chelates, which are then
reduced by a root FeIII-chelate reductase (EC 1.6.99.13)
encoded by the fro2 gene in A. thaliana[15]. The resulting
FeII is finally taken up by the plant at the level of the root
subapical zone by specific transporters belonging to the iron
regulated transporter (IRT) [3,5] and likely to the natural
resistance associated macrophage protein (Nramp) families
[2,20]. The transcript abundance from all the genes mentioned above increases in response to iron deficiency, although the molecular and cellular mechanisms involved in
this inducible integrated process remains to be worked out.
These plant iron deficiency responses are dependent upon
the genotype considered. This has been well documented in
the case of rice and tobacco plants, which were genetically
modified in order to over-express the Fe storage protein
ferritin [6,22]. Such a transformation led to Fe overaccumulation in various plant organs. This over-loaded Fe is
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G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
Table 1
Leaf fresh weight (FW expressed in g per plant), iron concentration (expressed in µM g–1 dry weight (DW)) and chlorophyll A + B concentration (expressed in
mg g–1 FW) of control A1 and transformed P6 and C5 genotypes of tobacco cultivated for 7 d in a solution containing 50 or 5 µM Fe-EDTA, and 0.5 or 2.0 mM
P. Data are mean ± S.D. of four different plants. Letters indicate values significantly different as measured by a t-test at the level of P < 0.05. Letters in subscript
correspond to the comparison between genotypes for a given treatment, letters in superscript to the comparison between treatments for a given genotype
0.5 mM P
Leaf fresh weight (g per plant)
Leaf Fe concentration (µM g–1 DW)
Leaf chlorophyll A+B concentration (mg g –1 FW)
A1
P6
C5
A1
P6
C5
A1
P6
C5
50 µM Fe
2.53 ± 0.64aa
2.19 ± 0.55aab
1.71 ± 0.06aa
0.73 ± 0.13ba
1.38 ± 0.12aa
1.43 ± 0.19aa
0.98 ± 0.07aba
1.00 ± 0.13aa
0.80 ± 0.09ba
likely to be sequestred within the over-expressed ferritin, and
would be, therefore, unavailable for plant metabolism. Consequently, ferritin transformed plants behave as Fe deficient
and have their root ferric reductase activated. Recently, Vansuyt et al. [21] have confirmed with tobacco that transgenic
plants over-expressing ferritin cultivated on sewage sludgeamended soils took up and accumulated more Fe than control
plants. Moreover, these authors showed that an addition of
phosphate in the culture medium increased the leaf Fe concentration in the control plant, abolishing the advantage conferred to transgenic plants by ferritin over-expression. These
data suggest a link between P and metal nutrition, which
could be physiological as previously shown by Huang et al.
[8] for Zn nutrition, rather than physico-chemical.
In this work, we compared the effect of Fe deficiency and
high P availability on the reductase activity, H+-ATPase activity and proton release by plants, by using previously described transgenic tobacco plants over-expressing ferritin
either in the plastids or in the cytoplasm [22]. Reductase and
H+-ATPase activities were determined on the plant roots,
while proton release by roots was determined by videodensitometry of pH dye indicator to quantify the intensity of
H+-fluxes and to locate the regions where the roots acidify
their environment. The H+ release patterns are discussed in
relation to biochemical measurements of root reductase and
H+-ATPase activities performed under the same conditions.
2. Results
2.1. Growth, and iron and chlorophyll concentrations in
leaves
The effect of Fe deficiency and high P availability on
growth and physiological status of the tobacco genotypes
were determined in cultivating plants for 7 d in solutions
containing 5 or 50 µM FeIII-EDTA and 0.5 mM P, or 50 µM
FeIII-EDTA and 2.0 mM P. Table 1 shows the biomasses, and
iron and chlorophyll concentrations in leaves of control (A1)
and transgenic tobacco plants over-expressing ferritin in the
5 µM Fe
1.74 ± 0.34aa
1.35 ± 0.18ab
1.19 ± 0.14aa
0.40 ± 0.04bb
0.57 ± 0.08ab
0.71 ± 0.04ac
0.72 ± 0.08ab
0.69 ± 0.08ab
0.43 ± 0.04bb
2.0 mM P
50 µM Fe
1.98 ± 0.24aa
2.28 ± 0.31aa
1.74 ± 0.08aa
1.01 ± 0.11ba
1.34 ± 0.10aa
0.97 ± 0.09bb
0.98 ± 0.18aa
0.87 ± 0.14a ab
0.75 ± 0.09aa
plastids (P6) or in the cytoplasm (C5). The statistical analysis
reveals that the nutrient conditions did not affect the biomass
production of C5 plants when compared to A1 control plants.
On the other hand, it was observed that the biomass production of the P6 genotype was decreased under Fe deficient
conditions (5 µM FeIII-EDTA and 0.5 mM P) comparatively
to Fe sufficient (50 µM FeIII-EDTA and 0.5 mM P) and P
abundant (50 µM FeIII-EDTA and 2.0 mM P) conditions. For
each nutrient conditions, no difference between genotypes
was observed.
In contrast, the nutrient conditions changed Fe concentration in leaves of different genotypes. Statistical analysis
indicates that, in a general manner, both the over-expressing
ferritin P6 and C5 genotypes had a leaf Fe concentration
higher than the one of control A1 genotype: 2-fold higher
under Fe sufficient (50 µM FeIII-EDTA and 0.5 mM P) and
1.5-fold higher under Fe deficient conditions (5 µM FeIIIEDTA and 0.5 mM P). However, Fe deficient conditions
(5 µM FeIII-EDTA and 0.5 mM P) decreased strongly the leaf
Fe concentration in all genotypes. The increase of P concentration from 0.5 to 2 mM also decreased the leaf Fe concentration in the C5 genotype, at a level similar to the one of the
A1 control genotype. As previously shown in soils for the C5
genotype, high P availability abolished the advantage conferred to transgenic tobaccos over-expressing ferritin for
increasing leaf Fe concentration, but an addition of KCl
instead of KH2PO4, in the medium did not change leaf Fe
concentration [21].
Leaf chlorophyll concentration also changed with nutrient
conditions : it decreased in all genotypes under Fe deficient
conditions. Moreover, the over-expressing ferritin C5 genotype had leaf chlorophyll concentrations significantly lower
than the ones of others, A1 control and P6 genotypes, when
supplied with 0.5 mM P.
2.2. Reductase and plasmalemma H+-ATPase activities in
roots
Table 2 shows the ferric reductase and H+-ATPase activities of tobacco plants grown under the same culture condi-
G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
29
Table 2
Root ferric reductase activity (expressed in nkat FeII per g FW) and H+-ATPase activity (expressed in kat P per g protein) of control A1 and transformed P6 and
C5 genotypes of tobacco cultivated for 7 d in solution containing 50 or 5 µM Fe-EDTA, and 0.5 or 2.0 mM P. Data are mean ± S.D. of four different plants for
reductase activity, and three root samples of 50 plants for ATPase activity. Letters indicated values significantly different as measured by a t-test at the level of
P < 0.05. Letters in subscript correspond to the comparison between genotypes for a given treatment, letters in superscript to the comparison between treatments
for a given genotype
0.5 mM P
50 µM Fe
Root ferric reductase activity II (nkat Fe per g FW)
Root H+-ATPase activity (kat P per g protein)
A1
P6
C5
A1
P6
C5
tions. Under Fe sufficient conditions, P6 and C5 ferritin
over-expressor genotypes of tobacco exhibited a significant
higher reductase activity comparatively to the one of A1
control plants, the P6 genotype having the highest. The
decrease of Fe concentration in solution from 50 to 5 µM
increased the ferric reductase activities of both P6 and C5
genotypes, but did not change the one of A1 control genotype. The increase of P concentration in solution from 0.5 to
2 mM had the same effect, even with the A1 control genotype, leading all the genotypes to exhibit a ferric reductase
activity as high as the one observed under Fe deficient conditions.
Under Fe deficient conditions, the root plasmalemma H+ATPase activities of both P6 and C5 genotypes were higher
than the one of A1 control plants, the C5 genotype having the
highest. The decrease of Fe concentration in solution increased the H+-ATPase activity of A1 and P6 genotypes at the
level of the one observed for the C5 genotype. The increase
of P concentration from 0.5 to 2 mM had the same effect than
the decrease of Fe concentration from 50 to 5 µM, and the
H+-ATPase activities of the three genotypes were equal and
similar to the one of the C5 genotype.
2.0 mM P
5 µM Fe
b
0.91 ± 0.37b
1.51 ± 0.50ab
1.42 ± 0.71abb
0.78 ± 0.20bc
1.74 ± 0.46bb
2.69 ± 0.44aa
50 µM Fe
ab
1.00 ± 0.42b
2.46 ± 0.40aa
2.40 ± 0.40aa
2.69 ± 0.46a a
2.93 ± 0.50aa
3.30 ± 0.60aa
1.73 ± 0.25aa
1.74 ± 0.41aab
2.06 ± 0.31aab
3.06 ± 0.43aa
3.23 ± 0.48aa
3.63 ± 0.53aa
a H+ release in the first 5 mm of the roots, of about
+3 pmol m–1 s–1, and an OH– release in the mature part of the
roots, which increased until –8 pmol H+ per m per s at 40 mm
from the root tips. There was no difference between the
genotypes.
When P was increased from 0.5 to 2 mM, the various
genotypes analyzed showed different H+-fluxes patterns. The
A1 control and P6 genotype had a uniform flux all along the
roots, with slight intensity of about –2 pmol m–1 s–1 for A1
and a null intensity for P6 genotype. In contrast, the C5
ferritin over-expressor genotype released H+ in the first
15 mm of the roots, with an intensity which arose
+7 pmol m–1 s–1. In the mature part of the roots, the C5 plants
released a slight flux of OH– of about –1 pmol H+ per m per s.
2.3. Proton fluxes released by roots
The effect of Fe deficiency and high P availability on the
release of protons by the tobacco genotypes were determined
by pH-indicator videodensitometry [9,14] of plants cultivated in solutions containing 5 or 50 µM FeIII-EDTA and
0.5 mM P, or 50 µM FeIII-EDTA and 2.0 mM P. The 4-weekold tobacco plants had six to 10 fine roots 60–90 mm long,
with few and short secondary roots which allowed H+-flux
measurements on isolated roots along 40–70 mm from the
root tip (Fig. 1). In general, the H+-flux released by the roots
was low, with absolute values less than 5 pmol H+ per m root
per s.
Under Fe sufficient conditions, the roots released OH–
anywhere (Fig. 2). The H+-flux in the first 5 mm of the roots
was weak, with about –3 pmol m–1 s–1, and stronger in the
mature parts of the roots, with about –7 pmol m–1 s–1. There
was no difference between the various genotypes analyzed.
Under Fe deficient conditions, the profiles of H+-flux showed
Fig. 1. Photograph of an agarose gel film containing bromocresol purple
after the root system of a tobacco plant was laid for 2 h for the measurement
of the proton flux released by the roots. The plant was a transformed C5
genotype cultivated for 7 d in a solution containing 5 µM Fe-EDTA and
0.5 mM P. The gel appears yellow in the regions where the root has acidified
its environment.
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G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
Fig. 2. Profiles of proton flux (expressed in pmol H+ per m root per s) released by the A1 control and transformed P6 and C5 genotypes of tobacco cultivated for
7 d in a solution containing 50 (Fe sufficient) or 5 µM (Fe deficient) Fe-EDTA, and 0.5 or 2.0 mM P (P abundant). For each treatment, the number of plants tested
comprised between two and four, and the number of roots measured between four and eight.
This C5 pattern was similar to the H+-flux profiles measured
for the three genotypes when plants were cultivated under Fe
deficient conditions and 0.5 mM P in the culture medium.
3. Discussion and conclusion
Ferritin over-expression in P6 and C5 genotypes has been
reported to increase Fe concentration in leaves resulting from
a storage of Fe in the ferritin protein [22]. However, the
storage of Fe in the ferritin protein induces a physiological Fe
deficiency, which is accompanied by related physiological
responses such as a decrease in chlorophyll concentration
and an increase in ferric reductase. Our results confirmed this
general pattern (cf. Tables 1 and 2): under Fe sufficient
conditions, the Fe concentration in the P6 and C5 genotypes
was double than the one of the A1 control genotype, the
chlorophyll concentration of C5 genotype was lower, and the
ferric reductase activity of both P6 and C5 genotypes was
higher than the one of the A1 control genotype. Furthermore,
the H+-ATPase activities of C5 and P6 plants were also
increased when compared to such activity in A1 control
plants. Increased H+-ATPase activity is known to be an indicator of iron deficiency, as is ferric reductase activity [16].
This observation is, therefore, consistent with the fact that
tobacco plant over-expressing ferritin, although having an
increased leaf Fe concentration, behaved as iron deficient.
However, these high enzymatic activities did not change the
flux of H+ released by roots in the medium.
The decrease of Fe concentration from 50 to 5 µM in
nutrient solution induced a Fe deficiency in all the genotypes:
decrease of Fe and chlorophyll concentrations in leaves,
increase of H+-ATPase activities in A1 control and P6 genotypes until raising the level observed with the C5 genotype,
increase of ferric reductase activities in P6 and C5 genotypes.
Moreover, in the three genotypes, these high enzymatic activities were accompanied by an increase of the flux of H+
released by roots in the medium at the level of the root
subapical zone, a characteristic response of dicot plants to
environmental Fe deficiency [16].
In addition, our results show that the increase of P concentration from 0.5 to 2 µM in nutrient solution had an intermediate effect between over-expressing ferritin and Fe deficiency. In A1 control and P6 genotypes, P concentration
increase did not affect the Fe and chlorophyll concentrations
in leaves, but increased strongly the H+-ATPase activity (× 2
and × 4, respectively) and the reductase activity (× 2) in A1
control genotype. However, these high enzymatic activities
were accompanied by an increase of the flux of H+ released
all along the roots. With C5 genotype, an increase in P
concentration in the culture medium induced, moreover, a
decrease in Fe concentration in leaves and an increase in flux
of H+ release preferentially at the level of the subapical
region of the roots. With the C5 genotype only, a P concentration increase induced a response similar to the one induced
by environmental Fe deficiency.
G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
Such observations raise several questions. A first question
is: why a high H+-ATPase activity did not induce in each case
a high flux of protons released by roots? The fact that a high
H+-ATPase activity did not correlate in each case with protons released by roots could simply be due to the fact that,
under these conditions, additional regulations could modulate H+-ATPase activity in vivo: these regulations would be
lost during the plasmalemma purification procedure, necessary to measure H+-ATPase activity in vitro. Alternatively,
this discrepancy between H+-ATPase activity and proton flux
could be due to a concomitant change in mineral ion uptake.
The net flux of protons released by a plant results from the
exchanged ion balance, mainly mineral cations and anions
taken up, and also organic anions excreted [7]. At the scale of
the whole plant, it thus results mainly from the needs of the
plant in mineral elements, and from their form and availability in the culture medium [4]. The H+-ATPase activity is thus
only one element among others involved in the establishment
of the proton release by plants.
Our results show, however, that a high flux of protons
released by roots at the level of the subapical region is
systematically associated to a low Fe concentration in plants
with regards to the sufficient conditions: 55, 41 and 50% in
Fe deficient conditions in regard to Fe sufficient conditions
for A1, P6 and C5 genotypes, respectively, and 68% for the
C5 genotype in P abundant conditions. A low Fe concentration in plants would also be needed to induce a high flux of
protons. The observation that a high flux of protons is released by roots at the subapical level indicates that this
activity takes place at the same root territory than the ferric
chelate reductase and the iron uptake activities. This strongly
suggests that the iron deficiency sensing system and signaling pathway regulating this territory-specific expression are
common to these three activities, i.e. acidification, reduction
and transport.
A second question is: how can a high P concentration in
solution induce a plant response similar to Fe deficiency?
The P concentrations used in this study were very high, and
likely generated chemical interactions with Fe in solution.
The P and Fe speciation at equilibrium in solution had been
calculated using Soilchem software [18]. The calculation
indicated that, at pH 6.3, 98–99% of P is bound with Ca, and
this ratio does not change significantly with P or Fe concentrations. Moreover, 56 and 4% of Fe is bound with EDTA,
and 44 and 96% with hydroxyls, for 5 and 50 µM Fe concentrations, respectively. The calculation shows therefore that, at
pH 6.3, the Fe concentration modifies the Fe speciation, but P
concentration has no significant effect on neither Fe nor P
speciation in solution. The interactions between Fe and P
become significant at lower pH.
Interactions between Fe and P could occur locally, at the
level of the cell walls which can be more acidic than bulk
nutrient solution, as suggested by several authors for Zn and
Cd in Arabidopsis halleri[10,27]. Such an environmental
interaction would induce an immobilization of P and Fe in
the cell walls, and consequently an environmental induced Fe
31
deficiency. Then, the Fe concentrations in leaves of A1 control and P6 over-expressing ferritin genotypes of tobacco
were not decreased under abundant P nutrition (see Table 1),
while ferric reductase and H+-ATPase activities were very
high. Only the C5 genotype showed a decrease in leaf Fe
concentration and increases in both enzymatic activities (see
Table 2). The results suggest, therefore, that Fe was not
immobilized in the cell walls, or if that happened, that this
decrease in local Fe concentration did not affect significantly
Fe uptake and accumulation by plants.
The last hypothesis was that such an effect could be
attributed, at least in part, to an activation of the root Fe
uptake system by P. It has been reported by Huang et al. [8]
that the tight control of P uptake by the P status of the plants
is lost under Zn deficiency, leading to very high accumulation
of P in plants. This effect was shown to occur at the gene
expression level since the transcript abundance of P transporters was increased under Zn deficient conditions. On the
other hand it is now well established that in iron deficient
Arabidopsis, Zn uptake can occur through IRT1, the major
root iron transporter [24], revealing a link, at the transport
level, between Zn and Fe. It is, therefore, reasonable to
postulate that P could regulate the root Fe uptake system at
the gene expression level. Also, and as already observed [21],
raising the P concentration from 0.5 to 2 mM in solution
leads to a leaf Fe concentration in the A1 control plants
similar to the one observed with the ferritin over-expressors.
In order to test this hypothesis, future experiments will be
performed with Arabidopsis for which (a) molecular probes
are already available and (b) ferritin over-expressors are
being constructed.
Our objective was to compare biochemical data (root
FeIII-chelate reductase and H+-ATPase activities) fluctuations in response to genetic modifications (ferritin overexpression) and environmental variations (Fe and P nutrition), with resulting phenomenological behavior at the whole
root system level (H+-flux). A good correlation of the data
obtained by the two types of approaches was obtained with
the three A1 (control wild type plants), P6 (which overaccumulates the Fe storage protein ferritin in the plastids),
and C5 (which over-accumulates the Fe storage protein ferritin in the cytoplasm) genotypes. Our study reveals that the
global measurement of H+-ATPase activity has some limits
compared to the localized determination of the variations of
H+-flux by videodensitometry all along the root. These limitations are likely due to the fact that part of the proton flux is
not directly linked to H+-ATPase activity. Nevertheless, the
combination of these various approaches enabled to demonstrate that the expression of “ferritin over-expression” character is largely dependent upon nutrition conditions, iron
nutrition of course, but also phosphorus nutrition. This
should be taken into account for the potential biotechnological use of such plants, in order to increase the iron concentration of various crop organs [6].
32
G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
4. Methods
4.1. Plant growth conditions
The plant material was tobacco (Nicotiana tabacum cv:
SR1), a control cultivar transformed with a cauliflower mosaic virus (CaMV) 35S promoter-beta-D-glucuronidase (EC
3.2.1.31) (GUS) construct (A1), and two transgenic cultivars
over-accumulating the Fe storage protein ferritin in the plastids (P6) or in the cytoplasm (C5). The cultivars were obtained as described by van Wuytswinkel et al. [22]. The
nutrient solution was a Hoagland solution containing 5 mM
KNO3, 5 mM Ca(NO3)2, 2 mM MgSO4, 50 µM H3BO3,
50 µM ethylene-diamine-tetra-acetic acid ironIII sodium salt
(Fe-EDTA), 50 µM MnSO4·H2O, 15 µM ZnSO4·7 H2O, 3 µM
Na2MoO4·2 H2O, 2.5 µM KI, 50 nM CoCl2·7 H2O and 50 nM
CuSO4·5 H2O. The solution pH was 6.3. Plant seeds were
germinated in vitro on a gel made with the nutrient solution,
1.0 g l–1 gelrite (Sigma G1910, Saint Louis, MI) and 0.6 g l–1
phytagel (Sigma P8169, Saint Louis, MI). After 7 d, the
plantlets were transferred for 14 additional days into glass
pots (five plants per pot) and placed on nutrient gel of the
same composition. The plantlets were grown in a culture
chamber with a 16-h light:8-h dark cycle, light intensity of
100 µmol photons per m2 per s and a temperature of 23 °C.
When the plants were 3-week-old, they were transferred to a
culture chamber with a light intensity of 300 µmol photons
per m2 per s and 75/80% relative humidity. The plants were
cultivated for 7 d more in hydroponic (30 plants per 12.5 l
pot) on nutrient solutions containing 5 or 50 µM FeIII-EDTA,
and 0.5 or 2.0 mM P added as KH2PO4. The solutions were
renewed twice during the culture period. The 4-week-old
plants were collected and used for measurements.
4.2. Determination of chlorophyll and iron concentrations
in leaves
The chlorophyll concentration in leaves was determined
by spectrophotometry at double optical wavelength (665 and
649 nm) [23]: 100 mg of mature fresh leaves were added to
10 ml cold acetone 80% (v/v), grinded in a Polytron mixer,
stored overnight at –20 °C, then centrifuged and diluted for
measurement. The measurements were repeated on two
leaves from three different plants. The leaf Fe concentration
was determined by spectrophotometry at 535 nm, using bathophenantrolin as FeII chelator, after reduction with thioglycollic acid of the acid hydrolysate samples [11]. Dry biomasses of roots and leaves were measured after drying at 60 °C
for 72 h. The measurements were repeated on four different
plants.
4.3. Determination of reductase activity in roots
The ferric reductase (EC 1.6.99.13) activity was measured
on the whole plants by spectrophotometry of the purple
colored FeII-ferrozine complex [26]. The plant roots were
immerged in a solution containing 0.1 mM FeIII-EDTA and
0.3 mM ferrozine (Sigma, Saint Louis, MI), and shaked with
a constant speed of 70 rpm min–1 in the dark at ambient
temperature. After 0, 10, 20 and 30 min, the solution was
sampled twice and the samples were analyzed by spectrophotometry at 562 nm using a molecular extinction coefficient of
28.6 mM–1 cm–1. The measurements were repeated on four
different plants.
4.4. Determination of plasmalemma ATPase activity in
roots
Plasma-membrane was purified from a root microsome
suspension by using the two phases partitioning system between polyethyleneglycol and dextran, then stored in liquid
nitrogen [25]. ATPase (EC 3.6.3.6) activity of plasmamembrane was measured as previously described by von
Wiren et al. [25]. It was inhibited by micromolar concentrations of vanadate (Ki = 8 µM), and was nearly fully dependent upon the presence of magnesium in the reaction medium. The vanadate sensitive ATPase activity accounted for
80% of total phosphohydrolase activity determined according to Santoni et al. [17]. Proteins were estimated according
to the procedure of Bradford [1]. The measurements were
repeated twice on three different samples, each one obtained
with 50 plants.
4.5. Determination of H+ release by roots
Profiles of H+-fluxes released by roots were determined
using pH-indicator dye videodensitometry [9,14]. The
method is based on following over time the evolution of
optical density images of a medium containing a pH indicator. Each image of optical density was converted first to
image of pH, secondly to image of total H+ concentration.
H+-fluxes per unit root length were then calculated as the
variations over time of total H+ amounts in the medium along
lines perpendicular to the roots. The variations of pH around
the roots were measured in films of agarose gel. Agarose
solution was prepared by adding 10 g of agarose powder
(Fluka 05068, Seelze, Germany) and 90 mg of bromocresol
purple (pK = 6.4) per liter of nutrient solution. The mixture
was boiled for 30 min, then cooled to 38 °C in a water bath
and its pH was adjusted to the same pH as the nutrient
solution, i.e. 6.3. The root system of the plants was positioned between two 100 × 200 mm2 rectangular sheets of
glass separated by 1 mm thick strips of PVC, and the agarose
solution was poured over. When the agarose was cooled to
ambient temperature, the upper glass sheet was removed
from the film of agarose gel to keep the root environment
unconfined. Two saturated calibration standards (basic and
acidic, respectively) were prepared for each series of measurements with the same culture solution, and acidified at pH
3.5 or alkalinized at pH 9.0 with HCl or KOH, respectively.
Measurements were made in a darkroom to avoid stray light.
However, the plants were illuminated between each image
G. Vansuyt et al. / Plant Physiology and Biochemistry 41 (2003) 27–33
capture. The images of the two standards were first acquired.
The images of the nutrient gel film containing the roots were
then acquired at 12 min intervals over a period of 2 h. The
scanned field was 124 × 200 mm2 and the images had 256 ×
256 pixels. All the images were acquired at the same magnification, giving a 0.50 × 0.81 mm2 area of gel film per pixel,
for a thickness of 1.0 mm. Images of pH and total H+
concentration were finally computed from the images of
optical density, and H+-fluxes along the roots were sampled
from the images of total H+ concentration as previously
described. The interval along the roots between two H+-flux
measurements was approximately the size of 1 pixel, i.e.
comprised between 0.50 and 0.81 mm. However, it was not
constant along a root, and it was different from one root to
another because of the different orientation of the roots in the
rectangular image grid. Each H+-flux profile measured along
a root was thus first regularized in space by linear interpolation. Then, means and standard deviations of H+-fluxes were
computed for each position along the root using the interpolated data of the different roots measured for each plant
treatment. The regular step kept was 1 mm. For each treatment, the number of plants tested comprised between two
and four, and the number of roots measured and used for
calculating each mean H+-flux profile comprised between
four and eight.
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
4.6. Statistical analysis
Statistical analysis were performed using the ANOVA
procedure of STATISTICA Software [19].
[18]
[19]
Acknowledgements
[20]
This work was supported in part by the “Programme
National de Recherche Sols et Erosion”.
[21]
References
[1]
[2]
[3]
[4]
[5]
[6]
M.M. Bradford, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principal of protein dye
binding, Anal. Biochem. 72 (1976) 248–254.
C. Curie, J.M. Alonso, M. Le Jean, J.R. Ecker, J.F. Briat, Involvement
of NRAMP1 from Arabidopsis thaliana in iron transport, Biochem. J.
347 (2000) 749–755.
C. Curie, Z. Panaviene, C. Loulergue, S.L. Dellaporta, J.F. Briat,
E.L. Walker, Cloning of ys1: an iron-regulated maize gene involved in
high affinity [Fe III] transport, Nature 409 (2001) 346–349.
R. Durand, N. Bellon, B. Jaillard, Determining the net flux of charge
released by maize roots by directly measuring variations of the alkalinity in the nutrient solution, Plant Soil 229 (2001) 305–318.
D. Eide, M. Broderius, J. Fett, M.L. Guerinot, A novel iron-regulated
transporter from plants identified by functional expression in yeast,
Proc. Natl. Acad. Sci. USA 93 (1996) 5624–5628.
F. Goto, T. Yoshihara, N. Shigemoto, S. Toki, F. Takaiwa, Iron fortification of rice seed by soybean ferritin gene, Nat. Biotech. 17 (1999)
282–286.
[22]
[23]
[24]
[25]
[26]
[27]
33
R.J. Haynes, Active ion uptake and maintenance of cation–anion
balance: a critical examination of their role in regulating rhizosphere
pH, Plant Soil 126 (1990) 247–264.
C. Huang, S.J. Barker, P. Langridge, F.W. Smith, R.D. Graham, Zinc
deficiency up-regulates expression of high-affinity phosphate transporter genes in both phosphate-sufficient and -deficient barley roots,
Plant Physiol. 124 (2000) 415–422.
B. Jaillard, L. Ruiz, J.C. Arvieu, pH mapping in transparent gel using
color indicator videodensitometry, Plant Soil 183 (1996) 1–11.
H. Küpper, E. Lombi, F.J. Zhao, S.P. MacGrath, Cellular compartmentation of cadmium and zinc in relation to other elements in the
hyperaccumulator Arabidopsis halleri,, Planta 212 (2000) 75–84.
S. Lobréaux, J.F. Briat, Ferritin accumulation and degradation in
different organs of pea (Pisum sativum) during development, Biochem. J. 274 (1991) 601–606.
H. Marschner, V. Römheld, Strategies of plants for acquisition of iron,
Plant Soil 165 (1994) 261–274.
G. Neumann, V. Römheld, Root excretion of carboxylic acids and
protons in phosphorus-deficient plants, Plant Soil 211 (1999)
121–130.
C. Plassard, M. Meslem, G. Souche, B. Jaillard, Localization and
quantification of net fluxes of H+ along maize roots by combined use
of pH-indicator dye videodensitometry and H+ selective microelectrodes, Plant Soil 211 (1999) 29–39.
N.J. Robinson, C.M. Procter, E.L. Connoly, M.L. Guerinot, A ferricchelate reductase for iron uptake from soils, Nature 397 (1999)
694–697.
V. Römheld, H. Marschner, Mobilization of iron in the rhizosphere of
different plant species, Adv. Plant Nutr. 2 (1986) 155–204.
V. Santoni, G. Vansuyt, M. Rossignol, Differential auxin sensitivity of
proton translocation by plasma membrane H+-ATPase from tobacco
leaves, Plant Sci. 68 (1990) 33–38.
G. Sposito, J. Coves, Soilchem: A Computer Program for the Calculation of Chemical Speciation in Soils, the Kearney Foundation of Soil
Sciences, University of California, Riverside and Berkeley, 1988.
Statistica édition 98, Kernel Version 5.1 M, StatSoft France
1984–1998, StatSoft Inc., Tulsa, Oklahoma.
S. Thomine, R. Wang, N.M. Crawford, J.I. Schroeder, Cadmium and
iron transport by members of a plant metal transporter family in
Arabidopsis with homology to Nramp genes, Proc. Natl. Acad. Sci.
USA 97 (2000) 4991–4996.
G. Vansuyt, M. Mench, J.F. Briat, Soil-dependent variability of leaf
iron accumulation in transgenic tobacco over-expressing ferritin,
Plant Physiol. Biochem. 38 (2000) 499–506.
O. van Wuytswinkel, G. Vansuyt, N. Grignon, P. Fourcroy, J.F. Briat,
Iron homeostasis alteration in transgenic tobacco over-expressing
ferritin, Plant J. 17 (1999) 93–97.
L.P. Vernon, Spectrophotometric determination of chlorophylls and
pheophytins in plant extracts, Anal. Chem. 32 (1960) 1144–1150.
G. Vert, N. Grotz, F. Dedaledechamp, F. Gaymard, M.L. Guerinot,
J.F. Briat, C. Curie, IRT1, an Arabidopsis transporter essential for iron
uptake from the soil and for plant growth, Plant Cell (2002)
1223–1233.
N. von Wiren, R. Gibrat, J.F. Briat, In vitro characterization of ironphytosiderophore interaction with maize root plasma membranes:
evidence for slow association kinetics, Biochim. Biophys. Acta 1371
(1997) 143–155.
Y. Yi, M.L. Guerinot, Genetic evidence that induction of root FeIII
chelate reductase activity is necessary for iron uptake under iron
deficiency, Plant J. 10 (1966) 835–844.
F.J. Zhao, E. Lombi, T. Breedon, S.P. MacGrath, Zinc hyperaccumulation and cellular distribution in Arabidopsis halleri, Plant Cell
Environ. 23 (2000) 507–514.