From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS UMR 5004, Agro-M/INRA, 34060 Montpellier Cedex 1, France
Received for publication, July 5, 2000, and in revised form, November 3, 2000
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ABSTRACT |
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In eukaryotic cells, ferritin synthesis is
controlled by the intracellular iron status. In mammalian cells, iron
derepresses ferritin mRNA translation, whereas it induces ferritin
gene transcription in plants. Promoter deletion and site-directed
mutagenesis analysis, combined with gel shift assays, has allowed
identification of a new cis-regulatory element in the
promoter region of the ZmFer1 maize ferritin gene. This
Iron-Dependent Regulatory Sequence (IDRS) is responsible for
transcriptional repression of ZmFer1 under low iron supply
conditions. The IDRS is specific to the ZmFer1 iron-dependent regulation and does not mediate the
antioxidant response that we have previously reported (Savino et
al. (1997) J. Biol. Chem. 272, 33319-33326). In
addition, we have cloned AtFer1, the Arabidopsis
thaliana ZmFer1 orthologue. The IDRS element is conserved in the
AtFer1 promoter region and is functional as shown by
transient assay in A. thaliana cells and stable
transformation in A. thaliana transgenic plants,
demonstrating its ubiquity in the plant kingdom.
Metals are essential for all living organisms but are toxic at
high concentrations. Therefore, metal homeostasis requires regulatory
circuits to coordinate metal uptake and storage (1). Such mechanisms
involve metal sensing proteins that transduce the signal within the
cell to modulate gene expression. These proteins often act as
regulatory factors, binding RNA or DNA cis-elements to
promote or repress gene expression (1, 2). Among these metals, iron is
one of the most abundant and is highly reactive as a pro-oxidant in
Haber-Weiss reactions to generate free radicals (3). Furthermore,
aerobic organisms have to overcome iron insolubility and then maintain
iron in a bioavailable form (4). Plants, due to their immobility, need
to tightly regulate iron homeostasis to prevent both iron toxicity or
deficiency (5). Recently, genes involved in iron uptake in plants have
been cloned: fro2, encoding a root ferric reductase (6), and
irt1, encoding a putative ferrous transporter (7). Inside
the plant cell, a particular class of plastid-localized proteins, the
ferritins, are able to store iron in a safe and bioavailable form (8). Ferritins are ubiquitous proteins found in bacteria, animals, and
plants, and, upon assembly, able to store up to 4500 iron atoms (4).
Ferritin gene regulation has become a model to study iron-regulated
expression both in the plant and animal kingdoms. In animal cells,
ferritin synthesis is mainly regulated at the translational level in
response to iron (2), whereas in plants transcriptional control has
been demonstrated in soybean cells (9). In maize, iron overload induces
accumulation of both ferritin mRNA and protein subunits (10, 11).
Two nuclear maize ferritin genes have been characterized and named
ZmFer1 and 2 (12). The ZmFer2 gene is
regulated by a cellular pathway involving the plant hormone abscisic
acid (ABA),1 whereas
ZmFer1 is regulated by an ABA-independent pathway (13). Using derooted plantlets and maize cell suspension cultures, we have
shown that this gene is regulated by both iron and redox signals (13).
Indeed, ZmFer1 mRNA increase of abundance is induced by
hydrogen peroxide treatments, and the iron-induced accumulation of this
transcript is abolished in the presence of N-acetylcysteine (NAC), an antioxidant agent. The accumulation of ZmFer1
mRNA induced by both H2O2 and iron
treatments were inhibited in the presence of the Ser/Thr phosphatase
inhibitors okadaic acid and calyculin A (13), suggesting the
involvement of dephosphorylation events in the transduction pathways
regulating ZmFer1 gene expression. To further characterize
the regulation of the ZmFer1 maize ferritin gene, we have
developed a transient expression assay in maize cells. Using this
system, a 2.2-kbp DNA fragment of the ZmFer1 gene was shown
to be sufficient to regulate reporter gene expression in response to
iron, with the same characteristics as observed for the endogenous
ZmFer1 gene regulation (13).
In this paper, this transient expression assay was used to identify an
Iron-Dependent Regulatory Sequence (IDRS) essential for the regulation
by iron of the maize ZmFer1 and the Arabidopsis thaliana AtFer1 ferritin genes. Stable transformation of A. thaliana plants was performed to demonstrate the functionality of
this element in planta.
Cell Cultures and Transient Expression Assays--
Maize BMS
(Black Mexican Sweet) cells were cultivated as described (13). A. thaliana cells (gift from Dr. Scheel) were cultivated at 24 °C
under constant shaking at 170 rpm, in 100 ml of MS medium (14),
containing 50 µM iron-EDTA. Subcultures were made every 2 weeks using 7.5 ml of cell suspension. For iron-starved cell cultures,
10 ml of 14-day-old cell culture were transferred to 90 ml iron-free
medium. Iron treatments were performed as described (13).
Eight-day-old iron-starved BMS or Arabidopsis cells were
transformed by particle bombardment as previously described (13). For
Arabidopsis cells the gun vacuum was set to 30 mbar and the helium pressure to 3.5 bars. The pRD109 and pAHC18 plasmids were used
as internal standards as described previously (13).
Gel Shift Assays--
Maize cell nuclear extracts were prepared
as described by Zhou et al. (15). Oligonucleotide sequences
used in these experiments are described below in Fig. 3B.
The DNA probe was end-labeled, and gel shift assays were performed as
described in Villain et al. (16).
Cloning of the AtFer1 Gene--
AtFer1 cDNA (17)
was used to screen an A. thaliana genomic library (18).
Positive bacteriophage DNA was prepared by polyethylene glycol
precipitation (19), and the genomic DNA fragment was subcloned in pUC18
and sequenced.
Plasmid Constructs--
All the constructs for transient
expression assays were made using standard recombinant DNA techniques
(19) and sequenced. Oligonucleotides are written in the 5' to 3'
orientation. An NsiI/SacI fragment containing
1600 bp of ZmFer1 promoter was subcloned into PstI/SacI-digested pRD109 to obtain p1600.
EcoRI digestion of p1600 and religation gave pPL. The
HindIII/NcoI fragment of p1600 was introduced in
pBI320X (20) to obtain p495. p124, p64, and p27 were obtained by
subcloning corresponding PCR fragments restricted with ClaI
and EcoRI into p495 previously cut with ClaI and
EcoRI. Primers used to generate PCR fragments were primers 1 (cccatcgatggacgcctccaccatccag) and 4 (gaattctgcgcgtgggtagtgg) for p124,
primers 2 (cccatcgatgctccctccgccttctcc) and 4 for p67, and primers 3 (cccatcgatgtctccaatctgctaccccc) and 4 for p27. To obtain p124luc the
ClaI/NcoI fragment from p124 plasmid was
subcloned in pSLluc+de. pSLluc+de was constructed by removing the
EcoRI site from the plasmid obtained after subcloning the
luc+ coding sequence from the pSP-luc+ vector (Promega) in the
EcoRI/NcoI-digested p59Luc (21). Site-directed
mutagenesis was performed to obtain pmTC1, pmTC2, and pmTC12, using the
Altered Sites II in vitro mutagenesis system (Promega) and
primers 5 (atatttctggatggccatggcgtccatcgatac) and 6 (gcggagggagcgcgccatgggctcgtggcggggc). Mutagenesis of the GC, SHS, G
box, and CAAT box sequences were made using the two-step PCR method as
described previously (22). The primers used were 1, 7 (ggtagcagattggagacccatggacggctgagatttggag), 8 (ctccaaatctcagccgtccatgggtctccaatctgctacc), and 4 to generate pmGb;
primers 1, 9 (gattggagacacgtggaccatggagatttggagaaggcgg), 10 (ccgccttctccaaatctccatggtccacgtgtctccaatc), and 4 to generate pmSHS;
primers 1, 11 (gagcgcgtggagggctcgccatgggcgggggactggggag), 12 (ctccccagtcccccgcccatggcgagccctccacgcgctc), and 4 to generate pmGC; and
primers 1, 13 (gcgtgggggtagcaccatggagacacgtggacggctgagat), 14 (atctcagccgtccacgtgtctccatggtgctacccccacgc), and 4 to generate pmCAATb.
All mutagenized promoter fragments were subcloned in the
ClaI/EcoRI-digested p124luc. AtFer1
PCR fragments were digested with EcoRI and NcoI
and subcloned in the pBI320X, using primers 17 (ccggaattcggatgtagcacgaggccgccac) and 15 (cgtggccatggttggaaaatgtagaagagg) for pIDRSWT; primers 18 (ccggaattcggatgtagcacgaggggatcccacggcccctacatc) and 15 for pmIDRS-1;
and primers 16 (ccggaattctttcatatccaccctccacg) and 15 for p Transgenic A. thaliana--
The constructs At1400IDRS and At1400
m*IDRS were excised by XhoI-SacI digestion and
subcloned into a pMOG 402 binary vector. The resulting plasmids were
introduced into Agrobacterium tumefasciens MP90 strain (23).
These bacteria have been used to transform A. thaliana using
the flower dip method (24).
Transgenic seeds were surface-sterilized and spread onto Petri dishes
containing MS/2 solid medium (14) and 50 µM iron-EDTA. Germination was synchronized by placing dishes for 2 days at 4 °C.
Subsequently, plants were transferred in a growth chamber under the
following conditions: 21 °C/18 °C, 16 h/8 h, day/night, 150 µE.s Analysis of ZmFer1 Promoter Deletions in Response to Iron--
In
a previous study (13), we have set up a transient expression
assay, using particle bombardment-mediated transformation, to study
ZmFer1 maize ferritin gene regulation. This system enabled us to show that a 2.2-kbp fragment of the ZmFer1 gene fused
to the Site-directed Mutagenesis of the ZmFer1 Promoter Region--
To
further analyze and localize the cis-acting elements
implicated in this regulation, a site-directed mutagenesis approach was
used. This approach was performed on a
Within the 124 bp of ZmFer1 promoter, some putative
regulatory sequences were identified by sequence comparison for
preferential mutagenesis as follows. A sequence named TC2 was found
only in the ZmFer1 promoter, at positions
The ZmFer1 promoter region contains a CAAT box sequence in
position
By a combined approach of promoter deletion and site-directed
mutagenesis, we have, therefore, been able to localize an essential region for ZmFer1 regulation by iron, which we have named
IDRS.
The IDRS Element Is Not Involved in the Antioxidant or Okadaic Acid
Inhibition of ZmFer1 Ferritin Gene Expression--
We have previously
shown, by Northern blot experiments and transient expression in maize
cells, that the iron-induced expression of ZmFer1 gene is
inhibited by NAC or OA (13). In the presence of NAC, the pSL5
ZmFer1-GUS fusion expression was strongly reduced in both
untreated and iron-treated cells, but an increase of GUS activity was
still induced by iron. Such results suggested that antioxidant agents
could affect the level of expression of this gene, rather than the
iron-dependent activation of ZmFer1 gene. Therefore, it was interesting to determine whether these effects are
mediated by the IDRS element. To address that question, p124Luc, pmTC12, and pmGC (see Fig. 2A) construct expression was
analyzed by transient expression in the presence of NAC or OA (data not shown). These experiments revealed that OA and NAC inhibitions of
ZmFer1 gene expression are not mediated by the IDRS, which appears to be specific to the iron response. Regulatory elements, distinct from the IDRS, are therefore required to mediate the antioxidant effect at the promoter level.
Characterization of the Iron-dependent Regulatory
Sequence--
To further characterize the IDRS element,
gain-of-function experiments were performed to establish whether this
sequence is sufficient to confer iron-dependent regulation
to a minimal promoter. A construct containing a
To determine if the IDRS element could effectively bind nuclear
transcription factors, gel shift experiments were performed. For this
purpose, a double-stranded deoxyoligonucleotide corresponding to the
IDRS was used (Fig. 3B). This
probe was incubated in the presence of nuclear extracts prepared from
maize cells treated or not by iron (Fig. 3A). In both low or
high iron conditions, only one complex was observed (Fig.
3A). The signal was more abundant after iron treatment, but
a signal was also clearly detectable when iron level was low (Fig.
3A, lane 2). Addition of 1 µM iron citrate in the protein extract from iron-depleted cells, or
The IDRS Element Is Involved in the Iron-regulated Expression of
the AtFer1 Ferritin Gene--
Maize is a graminaceous plant and
shares with this group some specificities in iron metabolism. A
phytosiderophore-mediated uptake of iron by the roots has been
described in this plant group, whereas it does not exist in
non-graminaceous monocotyledonous or dicotyledonous plants (32). This
raises the question of whether the IDRS element could also play a role
in iron-regulated expression in non-graminaceous plants. To address
that question, we have investigated the regulation of AtFer1
ferritin gene from A. thaliana, a dicotyledonous plant. It
has been previously shown, using AtFer1 cDNA as a probe
in Northern experiments, that this gene is regulated by an
ABA-independent pathway, and iron-induced accumulation of AtFer1 mRNA is inhibited in the presence of NAC (17).
These features match with ZmFer1 gene regulation and suggest
that AtFer1 would be the A. thaliana ferritin
gene orthologous to ZmFer1. We have cloned the
AtFer1 ferritin gene encoding the previously identified
AtFer1 cDNA (GenBankTM accession no AF229850). This gene contains an exon/intron structure similar to known plant ferritin
genes. A sequence comparison between the proximal region of
AtFer1 and ZmFer1 promoters is presented in Fig.
4A. This alignment reveals
three major blocks of identity. One corresponds to the Gbox sequence,
which is not involved in the iron-dependent regulation of
ZmFer1 gene (see above). A second region of similarity was revealed close to the putative TATA box of both promoters (Fig. 4A, block II). The third block corresponds to 14 bp with high similarity between the two promoters and corresponds to
the IDRS element. This region matches exactly with the
ZmFer1 IDRS base substitutions analyzed by gel shift (Fig.
3B), which affect the binding of nuclear proteins to the
IDRS (Fig. 3A). To determine whether this sequence
corresponds to a functional IDRS in the AtFer1 gene, a
transient expression assay was set up using particle bombardment-mediated transformation of Arabidopsis cell
suspension cultures. AtFer1 promoter deletions were fused to
the GUS reporter gene and introduced into cell cultures. The pIDRS-WT
construct contains 100 bp of the AtFer1 promoter and the
putative IDRS element. A 6-fold increase in GUS activity was measured
in the transient expression assay in response to a 500 µM
iron citrate treatment (Fig. 4). When the AtFer1 promoter
sequence was reduced from
Because stable transformation of A. thaliana plants is a
routine procedure, in contrast to maize transformation, we decided to
verify whether the IDRS was essential for the iron-mediated derepression of AtFer1 gene expression in transgenic
A. thaliana plants. The approach consisted of transforming
A. thaliana with 1400-bp AtFer1 promoter sequence
fused to the GUS reporter gene, wild type or mutated at the IDRS. The
effect of the mutation was evaluated by measuring the GUS activity in
transformed plants treated or not with iron. A 17-fold derepression of
the AtFer1-GUS fusion in response to iron treatment was
observed in the case of the wild type construct (At1400IDRS
in Fig. 5), whereas the IDRS mutation
(At1400 m*IDRS in Fig. 5) led to a derepression factor of
1.8-fold. This 9.5-fold decrease mediated by the IDRS mutation is due
to the increasing GUS activity in the iron-untreated plants, as
reported above in transient assays using shorter promoter sequences.
The GUS activity remains unaffected by the IDRS mutation in
iron-treated plants compared with the wild type construct. Similar
results were obtained by transient assay of the same constructs using
A. thaliana cell suspension cultures (data not shown).
Furthermore, as a negative control, a mutation of the block II sequence
(Fig. 4A) within the 1400-bp promoter was performed. This
mutation had no effect on the GUS expression, either in transgenic
plants or in transient cell assay (data not shown). Therefore, these
results, obtained in stable transformed plants as well as in transient cell assays, show that the IDRS is a major element of the iron regulated expression of the AtFer1 gene. They also indicate
that the IDRS effect can be similarly observed on a promoter sequence of 1400 or 100 bp (see pmIDRS2 in Fig. 4).
Using promoter deletion and site-directed mutagenesis analysis, we
have identified a cis-regulatory element, IDRS, essential for ZmFer1 maize ferritin gene iron regulation. This element
does not match with known regulatory sequences identified in plant promoters like the Metal Regulatory Element for example, nor with such
elements from yeast or animals (33). IDRS is required for iron-dependent regulation of both ZmFer1 and
AtFer1 genes, illustrating its broad functionality in the
plant kingdom. It is important to notice that the IDRS is not only
essential in transient assays using cell suspension cultures but also
in stable multicellular transformants such as transgenic A. thaliana plants (Fig. 5). Gain-of-function experiments failed to
demonstrate that IDRS is sufficient to confer
iron-dependent regulation, suggesting that other elements
are necessary. However, promoter sequence analysis of ZmFer1
and AtFer1 ferritin genes coupled to transient expression analysis showed that IDRS was the only conserved sequence essential for
iron regulation. Furthermore, the first evidence of iron
transcriptional regulation of a plant ferritin gene was obtained with
soybean (9). The corresponding gene was cloned (31) and contains an
IDRS element within its promoter at position Our results suggest that the IDRS element is involved in ferritin gene
repression at low iron concentrations. This could require a specific
chromatin conformation to allow IDRS bound protein(s) to interact with
the transcription initiation complex or to limit the TATA box access
for example. Then, if the IDRS fusion to the 35S CAMV minimal promoter
fails to reproduce such a correct conformation, the IDRS activity could
not be revealed.
In plant and animal cells, a common regulatory scheme of ferritin genes
by iron is emerging. Low iron conditions repress ferritin synthesis, at
the translational level in animals (2), and at the transcriptional
level in plants. In animals, a cis-element called Iron
Responsive Element (IRE), localized in the 5'-untranslated repeat of
ferritin mRNA, binds Iron Regulatory Proteins (IRPs). When iron
level is low, IRPs occupy IRE and prevent the initiation of mRNA
translation (2). Two types of IRPs, IRP1 and -2, have been
characterized. IRP1 enables integration on the IRE of at least two
different signals: the iron and the redox status of the cell. IRP1
binding is stimulated by iron deficiency or by H2O2 treatment. Iron could have a direct effect
on the protein, whereas the H2O2 signal would
require a transduction pathway (35). We have also described such an
integration of iron and redox signals for ZmFer1 maize
ferritin gene regulation (13). In this case, iron treatment promotes
ferritin synthesis, whereas antioxidant agents like NAC or glutathione
inhibit ferritin synthesis. However, in contrast to animal cells for
which such responses are targeted to the same molecule, IRP1, the redox
signal in plants is not mediated by the IDRS, which is specific to the
iron response. These results suggest that other(s) regulatory
sequence(s) in ZmFer1 mediate(s) the antioxidant response.
This integration of redox and iron signals within the ferritin promoter
could allow adjustment of the ferritin level and protection of the cell
according to the free iron concentration and the redox status of the cell.
IDRS implication in the transcriptional regulation of ferritin genes by
iron led us to search for this element in other gene promoter
sequences. Because iron uptake and storage need to be coordinated, some
iron-dependent regulatory mechanisms are required to
modulate gene expression implicated in these processes. In animal
cells, the IRE sequence regulating ferritin mRNA translation is
also localized in the 3'-untranslated repeat of the transferrin receptor mRNA (2). In low iron conditions, the binding of IRPs on
the IRE stabilizes the transferrin receptor transcript and allows the
synthesis of the corresponding protein. Therefore, the same regulatory
sequence enables the adjustment of iron storage and iron uptake
capacity of the cell. Searching for IDRS in plant sequence data bases
using BLAST and FASTA programs (36, 37), we have found no sequences
similar to IDRS in the promoter regions of genes that could be involved
in plant iron uptake, like fro2 (6), irt1 (7), or
the Nramp gene family (38, 39). Iron could also regulate
genes implicated in the protection against oxidative stress such as
superoxide dismutases or catalases. Again, we did not find the IDRS in
these genes. The A. thaliana genome is now almost completely
sequenced, and we have found some sequences similar to IDRS. However,
they were not localized in promoter regions, or were present in
unidentified sequences.
Gel shift experiments show that IDRS is able to bind nuclear protein(s)
in vitro, and these data correlate with in vivo
functional assays. The mechanistic significance of the apparent
increase of binding to the IDRS under iron overload remains to be
worked out. The molecular characterization of the protein(s)
interacting with the IDRS will allow the elucidation of the mechanisms
involved in the repression of ferritin synthesis during low iron
conditions, and the derepression by iron, of ZmFer1 and
AtFer1 ferritin genes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IDRS. To
generate pmIDRS-2, DNA fragment amplified with primers 19 (ccggaattccattggatgtagccatgggggatcccacggc) and 15, using pmIDRS-1 as
template, was subcloned in the
EcoRI/BamHI-digested pmIDRS-1. For the
gain-of-function constructs, the minimum 35S promoter was amplified
using primers (catcgatgacgtaagggatgacgcacaatcc and
cgaattctcctctccaaatgaaatgaacttcct) with p35Sluc as template and
subcloned in the ClaI/EcoRI-digested p124luc to
give pGFluc. Oligonucleotides
(cgacccgcccgccacgagccctccacgcgctat and
cgatagcgcgtggagggctcgtggcgggcgggt) were annealed and concatemerized.
Monomer, dimers, and trimers were selected and subcloned in the
ClaI-digested pGFluc. An AtFer1 1400-bp promoter
region was amplified using primers 15 and 20 (ccggaattcccatatctctcgagaaaggatagag), digested with EcoRI
and NcoI, and subcloned in pBI320X (pAt1400IDRS). To
generate the pAt1400m*IDRS, a two-step PCR approach was performed as
described (22) using primers 15, 20, 21 (ggggccgtgggatcccccatggctacatccaatgggatag), and 22 (ccggaattccattggatgtagccatgggggatcccacggc), and the PCR fragment was
subcloned in pBI320X.
1.m
2. After 10 days, plantlets were
transferred onto iron-free MS/2 solid medium for 4 days. Then, the
aerial parts of the plants were excised and incubated for 24 h
onto MS/2 liquid medium containing 500 µM iron-citrate or
no iron. Protein extracts were prepared, and reporter gene activity was
measured as previously described (13). Protein concentration was
determined according to the Bradford method (25).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucuronidase (GUS) reporter gene mimics the regulation of
the endogenous gene. An 8-fold increase of GUS activity was measured in
response to iron overload (13, see pSL5 in Fig.
1). Furthermore, iron-induced expression
of the ZmFer1-GUS fusion gene was inhibited by the
antioxidant agent NAC, and the Ser/Thr phosphatase inhibitor OA. These
results are in agreement with data obtained by Northern blot analysis
of the ZmFer1 gene expression. The ZmFer1-GUS
fusion pSL5 contained 1.6 kbp of the promoter sequence and the region
spanning from exon 1 to 3 (13). To localize cis-acting elements involved in the iron-dependent regulation of the
ZmFer1 ferritin gene, we have performed a deletion analysis
of the 2.2-kbp ZmFer1 fragment mentioned above. Intron
sequences were required to observe a significant level of expression of
the construct in maize cells (13). This was in agreement with our
general knowledge that introns are essential for the expression of many genes from monocotyledonous plants (26). These intron sequences act
mainly by stimulating the basal level of gene expression. However, it
could not be ruled out that intron sequences present in the pSL5
construct play a role in the iron-dependent regulation of
the fusion gene. To clarify this point, ZmFer1 intron
sequences were substituted by an intronic region of the rice actin
gene, which has been shown to strongly enhance reporter gene expression when present in a chimeric construct (27). Furthermore, we have previously demonstrated that this rice actin gene DNA fragment did not
contain iron-regulated sequences (13). This switch from maize ferritin
to rice actin intron sequences results in a strong increase of
expression of the construct in the absence of iron, although the
promoter sequence was the same (pSL5 and p1600 in Fig. 1). Iron
treatment of the cells lead to a 4.5-fold increase in GUS activity
(Fig. 1, p1600). This slight decrease from 8- to 4.5-fold of
the induction factor appears to be a result of the intron sequence
replacement. This phenomenon could be linked, in part, to the strong
stimulation of gene expression in the presence of the rice intron
sequences. However, the expression of the construct remains inducible
by iron overload, suggesting that essential sequences for this
regulation are not located within introns. The p1600 construct still
contains 61 bp of the ZmFer1 5'-untranslated region.
Deletion of this sequence up to the previously identified transcription
initiation sites (12) did not affect the pattern of expression of the
construct (data not shown). Therefore, a promoter deletion analysis was
performed, starting from the p1600 fusion gene containing 1600 bp of
ZmFer1 promoter sequence (Fig. 1). Reduction of the promoter
length from 1600 to 495 or 124 bp results in a decrease from 4.5- to
2.5-fold induction in response to iron (Fig. 1). Such a decrease could
be linked to the presence of regulatory sequences participating to the
iron-dependent regulation of ZmFer1 gene. These
deletions had no effect on the level of expression in low iron
conditions and demonstrate that essential iron response elements are
localized upstream of
124 bp. The reduction of promoter size from
124 bp to
64 bp lead to a more drastic effect. The expression level
in low iron conditions increased to the iron-induced level of the
124-bp construct, and stimulation by iron was abolished. A further
deletion from
64 to
27 bp did not change this pattern of
expression. The promoterless construct (pPL) does not support reporter
gene expression. Therefore, the 60-bp region spanning from
124 to
64 appears to contain essential regulatory sequences for iron-induced
expression of ZmFer1 maize ferritin gene. Furthermore, our
results suggest that this region would be involved in the repression of
ZmFer1 expression in low iron conditions.
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Fig. 1.
Effect of promoter deletions on the iron
stimulated expression of ZmFer1-GUS fusions in a
transient assay. Constructs correspond to a fusion between the GUS
reporter gene and deletions of the ZmFer1 gene promoter
fused to ferritin introns or rice actin introns. 8-day-old maize BMS
cells were cotransformed by two plasmids: a ZmFer1-GUS
fusion gene and the pAHC18 as internal control, and treated as
described previously (13). +1 corresponds to the first T of
the TATA box. Values are the mean of three independent transformations
from one representative experiment, out of at least three. The
error bars indicate standard deviations.
124-bp ZmFer1
promoter, which represents the minimal promoter to maintain an
iron-induced expression of the chimeric gene. This fragment was fused
to rice actin introns as mentioned above and to the luciferase reporter gene. A 3.3-fold increase in luciferase activity was measured in
response to iron treatment, when this construct was analyzed by
transient expression in BMS cells (Fig.
2B, p124Luc). This confirms the results obtained with the GUS gene as reporter (Fig. 1).
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Fig. 2.
Localization of an Iron-dependent
Regulatory Sequence by site-directed mutagenesis analysis of the 124
ZmFer1 promoter region. A, partial
nucleotide sequence of the
124-bp DNA fragment of ZmFer1
promoter. The putative regulatory sequences are boxed and
their names indicated. Stars indicate the mutated bases in
the various constructs used for transient expression (see
B). B, effect of promoter mutations on
iron-activated expression of the ZmFer1 gene in a transient
assay. White boxes represent the target sequences for
mutation. Black boxes represent mutated sequences. BMS cells
were transformed and treated as described in Fig. 1 except that the
plasmid used for luciferase activity standardization was pRD109, which
constitutively expresses the GUS reporter gene.
72 to
66, and
not in ZmFer2 promoter (Fig. 2A). These two
promoters share significant sequence identity in their proximal region
(12), but ZmFer2 is regulated by an ABA pathway and not by
the iron-dependent pathway studied in this paper (13). TC2
sequence mutagenesis led to a 3.3-fold increase of luciferase activity
under low iron conditions, and iron stimulation of expression was
reduced to a 1.4-fold increase (Fig. 2B, pmTC2).
The ZmFer1 promoter region contains another sequence similar
to the TC2 element, named TC1, localized at position
119 bp (Fig.
2A). TC1 sequence mutation alone did not affect the
iron-induced regulation of the reporter gene (Fig. 2B,
pmTC1). However, a double mutation of TC1 and TC2 resulted
in enhanced basal expression of Luc and absence of induction by iron
(Fig. 2B, pmTC12). These data suggests that, in
the absence of a functional TC2 element, the TC1 sequence could
partially substitute for TC2, whereas this sequence does not play a
major role in the wild type ZmFer1 promoter. Sequences
flanking the TC2 sequence were also analyzed to define more precisely
this putative cis-acting element. The region upstream of TC2
was of particular interest, because mutations in the sequence called GC
(Fig. 2A) lead to a pattern very similar to the TC2 mutant
(Fig. 2B, pmGC). This result extends the
regulatory region to both TC2 and GC sequences. This DNA fragment of 16 bp, which appears to be essential for the ZmFer1 ferritin gene regulation by iron, was named Iron-Dependent Regulatory Sequence (IDRS).
23 (Fig. 2A). This element has been described as
a general cis-acting element, but can also be involved in
specific responses. Indeed, ferritin H gene transcriptional control by
heme in Friend Leukemia cells is mediated by a CCAAT element (28), and
this sequence plays also a role in the transactivation of genes in response to antioxidant treatment in a human colorectal cancer cell
line (29). Mutation of the CAAT box sequence present in the
ZmFer1 promoter did not affect the expression pattern of the fusion gene in low or high iron conditions (Fig. 2). The Gbox element
(CACGTG) (Fig. 2A) is a common regulatory sequence in plant
gene promoters involved in many environmental stress responses (30).
Mutagenesis of the ZmFer1 promoter Gbox revealed this sequence not to be required for the iron-dependent
regulation of the ZmFer1 maize ferritin gene.
Transcriptional regulation of soybean ferritin gene has been clearly
demonstrated by nuclear run-on experiments (9). The corresponding gene
has been cloned (31), and we have compared the promoter sequences of
this soybean ferritin gene and ZmFer1. Interestingly, a
conserved region (Fig. 2A, SHS), distinct from
the IDRS element, was found in the proximal region of these two
promoters. However, mutational analysis of this sequence in a transient
expression assay revealed that this sequence is not involved in
ZmFer1 gene regulation by iron in maize cells (Fig.
2B, pmSHS).
80-bp 35S CAMV
promoter sequence fused to rice actin intron sequences, and the
luciferase reporter gene was prepared. The IDRS element was cloned
upstream of this construct, conserving the distance between this
regulatory sequence and the TATA box as in ZmFer1 gene. The
IDRS fragment corresponds to the ZmFer1 promoter region from
88 to
62 bp and was cloned in 1, 2, or 3 copies. For each
construct, a significant level of expression was measured, but no
iron-dependent regulation was observed (data not shown).
Increasing the fragment length to
124 to
62 of the ZmFer1 promoter led to the same result. These data suggest
that some additional elements are required to establish the iron
regulation or that a specific chromatin context is essential and not
reproduced in the fusion gene.
-mercaptoethanol as a reducer, or diamide as an oxidant in both
extracts, had no effect on the observed complex (data not shown). To
address the specificity of this complex, the reaction was performed in
the presence of a range of molar excess of cold probe. A
dose-dependent decrease of the binding was detected (Fig.
3A, lanes 4-7). In contrast, in the presence of
a 100-fold molar excess of nonspecific DNA, which did not contain IDRS,
no effect on DNA binding was observed. Therefore, a nuclear protein, or
protein complex, specifically binds the IDRS element in
vitro. Site-directed mutagenesis of the ZmFer1 promoter
enabled us to establish that base changes within IDRS affect the
chimeric ZmFer1-Luc gene regulation in vivo. To
investigate if such mutations alter the binding of nuclear proteins to
the IDRS in vitro, a competition experiment was performed in
the presence of different double-stranded oligonucleotides containing
three base substitutions compared with the WT sequence (Fig.
3B). When the binding reaction was performed in the presence of oligonucleotides M1 to M3, a partial inhibition of binding was
detected, but the complex was still clearly observed (Fig. 3A, lanes 9-11). Oligonucleotides M4 and M5
resulted in less inhibition of binding of nuclear protein(s) to the
labeled IDRS (Fig. 3A, lanes 12 and
13). These mutations correspond to the 3'-part of the IDRS,
the region mutated in pmTC2 construct in Fig. 2. Therefore, mutations
in this sequence lead to a loss of iron-dependent
regulation in vivo, and binding in vitro. These
experiments confirm that the IDRS is specifically bound by nuclear
protein(s) consistent with its in vivo ability to regulate
reporter gene expression in response to iron.
View larger version (63K):
[in a new window]
Fig. 3.
Specific binding of maize nuclear protein(s)
to the IDRS. Gel shift assays (A) were performed using
10 µg of nuclear extracts from BMS cells untreated or treated for
2 h with 5 mM iron citrate. Extracts were incubated
with 0.06 ng of double-stranded end-labeled IDRS probe. Competition
assays were performed with wild type or mutated unlabeled
oligonucleotides shown in B, or nonspecific unlabeled
oligonucleotides.
100 to
61 bp, deleting the IDRS, an
increase of GUS activity in low iron conditions was detected, and the
response to iron treatment was eliminated. This result is similar to
the data obtained by deleting the ZmFer1 IDRS (Fig. 1).
Using the pIDRS-WT construct, site-directed mutagenesis of the
AtFer1 IDRS was performed. In the pmIDRS-1 construct, the 3'
region of the IDRS was mutagenized, corresponding to the
ZmFer1 TC2 mutations (Fig. 2) and M4
M5 in
the gel shift assay. The complete IDRS was mutated in the pmIDRS-2 construct. Transient expression analysis of these plasmids revealed a
pattern similar to what is observed by the IDRS deletion. Site-directed mutagenesis of block II had no significant effect on the
iron-dependent regulation of the construct (data not
shown). Therefore, the IDRS is essential for the
iron-dependent regulation of both AtFer1 and
ZmFer1 ferritin genes, in transient assays.
View larger version (27K):
[in a new window]
Fig. 4.
Involvement of an Arabidopsis
sequence similar to ZmFer1 IDRS in
AtFer1 gene regulation in response to iron.
A, aligned nucleotide sequences of the ZmFer1 and
AtFer1 promoters. Stars indicate mutated bases in
the constructs used in transient expression analysis. B,
effect of promoter deletions and IDRS mutations on the iron-stimulated
expression of AtFer1-GUS fusions in a transient assay. The
white box represents the IDRS. Each cross
represents mutations of sequences similar to the GC or TC2 sequences of
the ZmFer1 gene as described in Fig. 2.
Arabidopsis cells were transformed as described in Fig. 1
except that the plasmid used for the standardization of GUS activities
was the p35Sluc.
View larger version (10K):
[in a new window]
Fig. 5.
IDRS-mediated iron-dependent
regulation of AtFer1-GUS fusion in A. thaliana transgenic plants. A. thaliana was
transformed using an AtFer1-GUS fusion containing 1400 bp of
promoter sequence mutagenized (At1400 m*IDRS) or not
(At1400IDRS) at the IDRS. Ten-day-old plantlets were starved
for iron for 4 days and then treated for 24 h in culture medium
containing 500 µM iron citrate or no iron. Protein
extracts were prepared, and GUS activities were measured. Derepression
factor corresponds to the ratio between GUS activities from
iron-treated plants to control untreated plants. Values correspond to
representative results out of three experiments on three independent
transgenic lines.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
121 to
108. Very
recently, it has been reported that the expression of a GUS reporter
gene fused downstream of this soybean promoter was repressed under low
iron conditions, in agreement with our findings (34). The soybean
promoter sequence suggested to be involved in this transcriptional
repression is different from the IDRS sequence. It is, however,
important to notice that this soybean regulatory region is not
conserved in AtFer1 and ZmFer1 promoter
sequences. Also, the result by Wei and Theil (34) does not exclude a
role of the soybean IDRS in iron control of ferritin gene expression in
this plant species.
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ACKNOWLEDGEMENTS |
---|
We acknowledge Dr. D. Scheel (IPF, Halle, Germany) for providing the A. thaliana cell line. We are grateful to Dr. Cathy Curie (Laboratoire de Biochimie et Physiologie Moléculaire des Plantes (BPMP), Montpellier, France) for giving the 35S-Luc and p59Luc plasmids. We are indebted to Dr. Frédéric Gaymard (BPMP, Montpellier, France) for providing the A. thaliana genomic library. We thank Céline Forzani (BPMP, Montpellier, France) and Dr. Tim Tranbarger (BPMP, Montpellier, France) for critical reading of this manuscript.
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FOOTNOTES |
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* This work was supported by the CNRS (Program Biologie Cellulaire: du Normal au Pathologique Grant 96003).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF229850.
Present address: Génétique Moléculaire des
Plantes, Université de Picardie Jules Verne, 80039 Amiens
Cédex, France.
§ To whom correspondence should be addressed: Tel.: 33-4-9961-2323; Fax: 33-4-6752-5737; E-mail: lobreaux@ensam.inra.fr.
Published, JBC Papers in Press, November 22, 2000, DOI 10.1074/jbc.M005903200
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ABBREVIATIONS |
---|
The abbreviations used are:
ABA, abscisic acid;
NAC, N-acetylcysteine;
OA, okadaic acid;
BMS, Black Mexican
Sweet;
MS, Murashige and Skoog medium;
GUS, -glucuronidase;
IRE, iron-responsive element;
IRP, iron-responsive protein;
IDRS, iron-dependent regulatory sequence;
CAMV, cauliflower
mosaic virus;
bp, base pair(s);
PCR, polymerase chain reaction;
SHS, soybean homologous sequence.
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