(Received for publication, August 2, 1994; and in revised form, January 23, 1995)
From the
We report the purification of the unstable aconitase enzyme from
melon seeds and the NH-terminal amino acid sequence
determination. Antibodies raised against this protein enabled the first
isolation and characterization of cDNA encoding aconitase in plants. A
full-length cDNA clone of 3210 base pairs was isolated from a library
of cDNA clones derived from immature pods of Arabidopsis
thaliana. The amino acid sequence deduced from the open reading
frame includes the sequence obtained by direct sequencing of the
NH
terminus of the purified enzyme. Genomic clones of the
aconitase gene were isolated, and comparison of the cDNA and genomic
sequences reveals that the coding sequence is divided among 20 exons.
There are five putative sites for transcription initiation. The
aconitase gene is constitutively expressed, but at a low level, during
most developmental stages, with a dramatic increase during seed and
pollen maturation and during germination. Surprisingly, plant
aconitases have reasonably high homology to binding proteins for
iron-responsive elements from mammalian species, opening the
possibility that a similar type of translational regulation occurs in
plants.
Aconitase, also known as citrate (isocitrate) hydrolyase or aconitate hydratase (EC 4.2.1.3) is an enzyme containing an Fe-S cluster that catalyzes the reversible isomerization of citrate to isocitrate via cis-aconitate in the tricarboxylic acid cycle.
This enzyme is found in two cellular compartments(1) . The mitochondrial aconitase is implicated in the Krebs cycle. The physiological role of the cytoplasmic aconitase is not well characterized and seems to be different between plants and mammals.
In animals, study of the mitochondrial aconitase has increased our
knowledge of the Fe-S cluster(2, 3, 4) . The
Fe-S cluster is implicated in the dehydration and hydration steps
during the enzymatic reaction. The labile iron (Fe),
spontaneously lost by oxidation during purification, is involved in the
binding of the substrate(5, 6) . Recently, Rouault et al.(7) have found as much as 56% homology between
porcine heart aconitase and iron-responsive element-binding protein
(IRE-BP). (
)The IRE-BP is an RNA-binding protein that plays
a regulatory role in the iron metabolism. Kaptain et al.(8) have demonstrated that IRE-BP also has an aconitase
activity. Thus in animals, cytoplasmic aconitase has a dual function:
(i) an IRE-BP activity involved in the post-transcriptional regulation
of iron metabolism(9, 10, 11) , and (ii) an
aconitase activity whose physiological role is not well understood.
These two activities are mutually exclusive. When the cluster is in the
4Fe-4S state, the protein has the aconitase activity but not the IRE-BP
function. Alternatively, when the cluster is in the 3Fe-4S state, the
cytoplasmic aconitase has the IRE-BP function but not the aconitase
activity. However, the mechanism involved in the transition between the
two forms is not understood although several models have been proposed (12, 13, 14, 15) .
Several studies have partially identified the amino acids of the IRE-BP and the nucleotides of the IRE (iron-responsive element, the stem-loop structure implicated in the binding of the IRE-BP) involved in the interaction(16, 17, 18) .
Aconitase has been less well studied in plants. Nevertheless, the mitochondrial aconitase has been purified from potatoes (19) and structural data obtained using the EXAFS (extended X ray absorption fine structure) technique have shown that the Fe-S cluster is different from the one observed in animals(20) .
In plants, cytoplasmic
aconitase and mitochondrial aconitase show similar characteristics and
cannot be differentiated(21) . On the other hand, in animals,
the two aconitases are easily separated by ion exchange chromatography (22) and are encoded by two different genes (23, 24) . Recent results, showing that plant
aconitase is inhibited by HO
(19) and
that no activity is detected in glyoxysomes(25) , have led the
authors to propose that plant cytoplasmic aconitase participates in the
glyoxylate cycle.
We report here the first isolation and characterization of a plant aconitase gene.
Figure 1: Purification of aconitase from melon seeds. Samples from the different steps of C. melo aconitase purification were separated by electrophoresis on a 10-15% gradient SDS-polyacrylamide gel and visualized by silver staining. Lane 1, molecular size markers (Bio-Rad); lane2, crude extract (0.5 µg); lane3, extract after ion-exchange chromatography using Q Fast Flow (0.5 µg); lane4, extract after affinity chromatography using Yellow 86 (0.5 µg); lanes 5 and 6, extract after hydrophobic interaction chromatography using Phenyl 5PW: fraction I (0.08 µg, lane5) and fraction II (0.08 µg, lane6); lanes7 and 8, extract after ion exchange chromatography using Mono Q: fraction I (0.06 µg, lane7) and fraction II (0.06 µg, lane8).
The cDNA clone was shown to be incomplete since the
NH-terminal sequence obtained by Edman degradation was not
found in the predicted amino acid sequence from the cDNA. This result
is not surprising since a 98-kDa protein is expected to be encoded by a
cDNA almost 3 kb long. In order to obtain a full-length cDNA, the fruit
melon cDNA library was screened twice with a 300-bp DraI
fragment present only in the longest cDNA. Seven new clones were
isolated, but they appeared to be identical to the longest clone
previously isolated.
In order to obtain a complete plant cDNA
encoding the aconitase enzyme, a cDNA library constructed from A.
thaliana pod RNA was screened under heterologous conditions with a
2.2-kb EcoRI fragment from the longest melon cDNA. After
hybridizing in 30% formamide at 42 °C followed by washing twice in
2 SSC at 42 °C for 30 min, seven positive clones were
isolated (three clones of 3.2 kb and four clones of 2.3 kb). No
difference was observed between clones of the same size after
restriction, hybridization, and partial sequencing. The longest cDNA
clone of 3210 bp was completely sequenced. The NH
-terminal
sequence obtained by Edman sequencing of the melon aconitase matched
the amino acid sequence predicted by the A. thaliana cDNA
sequenced, suggesting that the A. thaliana cDNA encodes the
aconitase enzyme. Fig. 2shows the homology between the
NH
-terminal amino acid sequence obtained from melon seed
aconitase, potato mitochondrial aconitase, (
)and the
sequence deduced from the A. thaliana cDNA. The sequence of
the cDNA contains an open reading frame that encodes 923 amino acid
residues, with a predicted molecular mass of 98,490 Da and a pI value
of 6.0. Three consensus polyadenylation sites AAUAAA (38) at
nucleotides 2809, 2940, and 3026 are present in the 3`-untranslated
region of 441 bp. No stop codon was found at the 5` end of the gene,
and in order to define the complete transcribed sequence we have
isolated the A. thaliana gene encoding the aconitase enzyme
from a library of genomic fragments.
Figure 2:
Homology alignment of the
NH-terminal sequence of aconitase from different plant
species. The three-letter code for amino acids is used
throughout. Residues common to the three plant species are boxed. Sequences shown are A. thaliana (protein
sequence predicted from A. thaliana cDNA sequence), C.
melo (protein sequence obtained from melon seed aconitase), and Solanum tuberosum (protein sequence obtained from potato
mitochondrial aconitase).
The mapping of these genomic clones by Southern blot analysis using the four EcoRI cDNA fragments (0.2, 0.8, 1.6, and 0.6 kb) showed the 3` end of the gene was absent as the 0.6-kb probe did not hybridize to the clones.
To obtain the missing 3` extremity of the aconitase gene, we
have screened a new genomic library (EMBL3) using the 0.6-kb probe. One
clone was isolated after screening 2 10
plaques.
Restriction analysis of this clone and Southern blot mapping enabled
the completion of the physical map of the aconitase gene of A.
thaliana (Fig. 3). The genomic clones were sequenced (6760
bp). A comparison between the genomic and the cDNA sequences of the A. thaliana aconitase shows that this gene is divided into 20
exons (Fig. 3). Table 2indicates the exon and intron
sizes and the intron-exon junction position. The exon-intron borders
match the criteria for consensus sequences of splice junctions, the
GT/AG rule(39) . A recent study shows that in plants there is
no conserved sequence for branch point selection for lariat formation
and that in dicotyledonous plants the introns are AT-rich(40) .
The introns of the A. thaliana aconitase gene are in agreement
with these observations. The three polyadenylation signals AATAAA
observed after cDNA analysis are localized at positions 5068, 5199, and
5285, respectively.
Figure 3:
Genomic organization of A. thaliana aconitase. A, partial restriction map of the
genomic clones isolated by screening the Dash and the EMBL3
libraries with the full-length cDNA. B, BamHI; E, EcoRI; H, HindIII; Ps, PstI; Pv, PvuII; X, XbaI. B, diagram of the gene structure (exons are
represented by filled boxes; introns are represented by opened boxes; 5`-noncoding region is represented by box
with diagonal lines; 3`-noncoding region is represented by box
with horizontal lines). C, partial restriction map of the
predicted cDNA.
In order to assess the complexity of the gene encoding the aconitase enzyme in A. thaliana, Southern blot analysis was carried out on genomic DNA isolated from two ecotypes of A. thaliana (Columbia and C24) and digested with several restriction enzymes. Filters were hybridized with the three EcoRI cDNA fragments (0.8, 1.6, and 0.6 kb) as a probe (Fig. 4). The physical map obtained is identical to the one observed for the genomic clones, suggesting the presence of a unique gene in A. thaliana.
Figure 4:
Southern blot analysis of A. thaliana genomic DNA for aconitase gene. Genomic DNA was isolated from two
ecotypes C24 (C24) and Columbia (Col) of A.
thaliana leaves and digested with the following restriction
enzymes: PvuII (Pv), EcoRI (E), BamHI (B), HindIII (H), PstI (Ps), SspI (Ss), and XbaI (X). Five µg of digested DNA was
fractionated on a 0.8% agarose gel and blotted onto Hybond-N membrane. The membrane was hybridized with EcoRI
fragments of A. thaliana cDNA.
Among the 31 sequenced clones containing an uncoded 5` terminal cap G residue, 14 clones stopped at position +1, 7 at position -2, 4 at position -4, 5 at position -9, and one at position +9. So, the precise mapping of the transcription start site has revealed five putative sites with one preferentially used that we have positioned at +1. We have also observed five clones without the additional G residue (one clone at position +14, 3 clones at position +20, and 1 clone at position +29). These clones most probably arise from the premature stopping of the reverse transcriptase. Finally, another clone without the additional G residue positioned at -49 was obtained. This clone is not an amplified DNA genomic fragment since a longer size would be expected due to the introns. Thus, the possibility that a second promoter exists cannot yet be ruled out.
The promoter region shows a classical TATA box positioned at -32, which is in agreement with the position given by Joshi(42) , i.e. 32 ± 7 bp from the transcriptional start site. Moreover, multiple transcriptional start sites are often observed in housekeeping genes(43) .
The first ATG occurs at position +91 relative to the transcriptional start site. This ATG may be the initiation codon because the surrounding sequences resemble the consensus sequence TAACAATGGCT(38) . There is continuous open reading frame from the transcriptional start site to the first methionine. It may be possible that this sequence encodes a mitochondrial leader peptide. However, an aspartic residue is found at position 25 and a glutamic residue at position 14. Acidic residues are normally absent from the mitochondrial transit peptide (44) , but there is not a strict consensus sequence(45) .
Figure 5:
Northern blot analysis of aconitase
expression in A. thaliana and B. napus organs. Total
RNA (50 µg/lane) from A. thaliana 6-day seedlings (lane1), leaf (lane2), stem (lane3), young flower before anthesis (lane4), mature flower (lane5), immature
pod (lane6), mature seed (lane7),
and from B. napus leaf (lane8), pistil (lane9), and anther (lane10) were electrophoresed in a denaturing 1.5%
agarose-formaldehyde gel and blotted onto Hybond-N membrane. The membrane was hybridized with the aconitase cDNA
clone.
Figure 6:
-Glucuronidase expression in A.
thaliana primary transformant organs. A 1339-bp
5`-HindIII/BglII promoter fragment of the A. thaliana aconitase gene was subcloned into a
-glucuronidase expression vector.
-Glucuronidase-expressing
cells are indicated by their blue color. PanelA is a light microscopic view of a mature flower. PanelB shows a microscopic view of a mature
anther.
There are only two previous reports of purification of plant aconitase. The first characterization of plant aconitase, from potato mitochondria, was made by Verniquet et al.(19) . The second (50) revealed three aconitase isoforms isolated from etiolated pumpkin cotyledons. The subcellular distribution of these purified aconitase isoforms was not clearly determined. Brouquisse et al.(21) found no difference between cytosolic and mitochondrial aconitase in plants. On the other hand, in animals the mitochondrial and cytosolic aconitases have different isoelectric points and are encoded by two different genes(22, 23) . In yeast, the mitochondrial and the cytoplasmic aconitases are encoded by a single gene(51) . After disruption of the yeast mitochondrial aconitase gene, no aconitase activity is detected in crude extracts. A single gene encoding two proteins with different subcellular locations has been described for yeast fumarase(52) , for yeast invertase(53) , and for yeast histidine and valine tRNA(54, 55) .
In plants, more work must be done to demonstrate if mitochondrial and cytoplasmic aconitases are encoded by a single or two distinct genes. Such a situation where a single gene encodes two transcripts has been observed in plants for petunia chalcone isomerase (56) and for zeins in maize seeds(57) .
During this study, we have
compared the molecular weight and the pI values of plant aconitase with
aconitase of different species and noted that they share similar
characteristics with the mammalian IRE-BP(58, 59) .
Two peaks of aconitase activity were detected during the purification
of aconitase from melon seeds, resembling the situation described by
Neupert et al.(58) , who found that purified human
placental IRE-BP had two major bands of 95 and 100 kDa. Purified cow
liver IRE-BP is composed of one or two bands on SDS-PAGE; the number of
bands is dependent on the reducing agent concentration(60) .
The heterogeneity observed for human placenta IRE-BP is localized on a
NH-terminal fragment obtained by proteolysis but not
necessarily in the first amino acid residues(59) . A similar
situation may occur here for the two aconitase enzymes isolated from
melon seeds. However, we cannot exclude other kinds of modifications
like proteolysis, post-translational modification, or nonenzymatic
deamidation.
The high degree of similarity observed between plant aconitase and IRE-BP is surprising. Until now IRE-BP activity has been detected in mammals, fishes, flies, and frogs but has not been reported in yeast, bacteria, and plants(61) . However, the IRE probes used in these gel retardation assays were from human ferritin messenger and from human transferrin receptor messenger. These two messengers are implicated in iron metabolism and are post-transcriptionally regulated by the IRE-BP (9, 10, 11) . In plant ferritin messengers, no IRE was found by homology with mammals IRE(62, 63, 64) . It is possible that plant aconitase is also implicated in a post-transcriptional regulation because it shares a high homology with mammalian IRE-BP. In order to show this, it is necessary to find out if IRE-like structures are present in plant messengers.
IRE-BPs are considered to be a new class of RNA-binding proteins because there is no significant homology between the IRE-BP and the RNA recognition motif identified in other RNA-binding proteins(65) . In the A. thaliana aconitase, we have observed a sequence (DLVIDYSVQV) similar to the one recently reported by Basilion et al.(16) (DLVIDH-IQV, corresponding to amino acids 121-130 in human IRE-BP). This sequence cross-links to the IRE. Nevertheless, it is necessary to identify other contact points between IRE and IRE-BP in order to understand the complete interaction.
We are interested in finding out if plant cytosolic aconitase is involved in a post-transcriptional regulation in an analogous fashion to the mammalian IRE-BP and if the plant mitochondrial and cytosolic aconitases are encoded by a single gene.
This article is dedicated to the memory of Dr. Jean-Louis Peyret, whose tragic death was a great loss to all of us.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X82839[GenBank]-X82841[GenBank].