Marc Vauterin and Michel Jacobs. Laboratory for Plant Genetics, Vrije Universiteit Brussel, Paardenstraat 65, 1640 Sint-Genesius Rode, Belgium. Received 21 ...
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Plant Molecular Biology 34: 233–242, 1997. c 1997 Kluwer Academic Publishers. Printed in Belgium.
Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase Val´erie Frankard , Marc Vauterin and Michel Jacobs Laboratory for Plant Genetics, Vrije Universiteit Brussel, Paardenstraat 65, 1640 Sint-Genesius Rode, Belgium Received 21 November 1996; accepted in revised form 13 February 1997
Key words: amino acid biosynthesis, Arabidopsis thaliana, monofunctional aspartate kinase, PCR cloning, threonine overproduction
Abstract A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product. Introduction In bacteria and plants, aspartate kinase (AK) catalyses the first step of the synthesis of essential amino acids lysine, threonine, isoleucine and methionine, through activation of aspartate to aspartyl-phosphate by ATP [6]. Due to both the complexity of the pathway and the specificities of the different bacterial species, structure and regulation of aspartate kinase have evolved in a very diverse manner. More specifically, in Escherichia coli, three distinct isofunctional AKs have been identified, encoded by genes thrA, metL and lysC, differing in the way their synthesis and activities are each regulated by the respective concentrations of one of the end products of the pathway. Furthermore thrA and metL code for a bifunctional protein, carrying homoserine The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X98873.
*135302*
dehydrogenase (HSDH) activity at the COOH side of the AK sequence. All three enzymes are homomers, but subunit size and number differ for each case [for review, see 11]. Bacillus subtilis also has three aspartate kinase isoforms, but effectors are meso-diaminopimelate and lysine alone or with threonine, and none are bifunctional [37]. The two lysine sensitive forms are heterodimers produced by in-frame overlapping genes lysC and lysC . Corynebacterium sp. possess only one AK regulated in a concerted manner by lysine and threonine, with the same quaternary structure as the Bacillus lysine-sensitive enzymes [22]. In fungi, lysine is not derived from aspartate but from -ketoglutarate via the aminoadipic acid (AAA) pathway, so that multiple forms of AK may not be necessary and regulation is not as complex as observed in most bacteria. To date, a single AK encoded by gene HOM3 has been demonstrated, for which threonine was shown to be both repressor and inhibitor [27].
GR: 201001909, Pips nr. 135302 BIO2KAP plan3755.tex; 7/05/1997; 13:26; v.7; p.1
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Figure 1. Nucleotide sequence and predicted amino acid sequence of the Arabidopsis thaliana ak-lys1 cDNA encoding monofunctional AK. The beginning of the cDNA is shown, as well as the KFGG box (1), the DP site (2). The position of the two primers used to amplify the original PCR fragment is underlined.
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235 The AK status in higher plants has not yet been fully uncovered. The presence of at least two isozymes has been demonstrated biochemically by assaying enzymatic activity in presence of potential feedback inhibitors: one inhibited by lysine and synergistically by lysine plus S-adenosylmethionine (the most abundant) [29], and the other by threonine (for review [17]). In a few plants including A. thaliana [20], part of the activity does not seem inhibitable by any effector, but the existence of an isozyme insensitive to feedback control as in E. coli has never unquestionably been shown. Modification of the feedback inhibition properties of either the lysine- or the threonine-sensitive isozyme results in overproduction of threonine in the free pool of amino acids, much more pronounced in the former case [3, 4, 9, 13, 14, 16, 19, 20]. Genetic studies based on such mutants in barley and maize have shown that two unlinked loci encode lysine-sensitive AKs [3, 14]. To date, molecular characterization of one class of AK isozyme has been reported in carrot [35], in Arabidopsis thaliana [18] and in maize [26]. Interestingly, this AK isozyme carries at the COOH end a second enzymatic activity for HSDH, the third step of the common pathway. The deduced plant amino acid sequences have highest homology with the E. coli thrA gene, coding for the bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase (AK-HSDH). Surprisingly however, the number of genes coding for this class of isozyme varies from one in A. thaliana to at least three in maize. This paper describes the isolation and characterization of a cDNA encoding a monofunctional AK, the first to be cloned and analysed from a plant. Evidence for the presence of a second ak gene is also presented, and thoroughly described in the accompanying paper. This duplication of expressed sequences is not an uncommon feature in plant, including the relatively small genome of Arabidopsis. Further investigation will be necessary to clarify their possible roles in relationship to developmental and environmental regulation.
Materials and methods Plant gene source Arabidopsis thaliana race Bensheim was used for Southern blot analysis and PCR amplification. The FIX genomic bank (Landsberg erecta) was kindly provided by Dr H. Goodman, Boston, USA. The
Figure 2. The cDNA and genomic banks were screened through hybridization with a radioactive PCR probe and through PCR amplifications with the same primers. One cDNA (around 300 bp) and three genomic clones were identified, among which two of different amplification product length (as indicated by the arrows, around 500 and 440 bp). Lane 1, ak-lys1 cDNA (pure); lanes 2, 3, 4, positive genomic clones (non-pure); lane 5, genomic DNA.
II cDNA constructed from poly(A)+ mRNA extracted from cell suspension cultures of race Landsberg erecta was kindly provided by Dr Trezzini (Max Planck Institut, Cologne, Germany).
UNIZAP
PCR amplification of ak-lys sequences PCR amplification of ak-lys fragments was done on A. thaliana genomic DNA in the following conditions for a total volume of 25 l: 1 l of DNA (0.5 g/l), 10 mM Tris-HCl pH 8.3, 1.5 mM MgCl2 , 50 mM KCl, 0.1 mg/ml gelatin, 0.2 mM each dNTP, 0.5 M each primer and 0.5 units Taq DNA polymerase (Boehringer, Mannheim). Reaction mixes were cycled 35 times: 94 C/50 C/72 C each step for 1 min. The concentration of the degenerated primers was optimized. The primers used to isolate the ak-lys fragments were: (1) degenerate primer in the sense orientation corresponding to the ‘lysine-box’, (TTLGRGGSDTTA), amino acids 193 to 204 (position 1 on K of KFGG): 50 -ACNACN(C/T)TNGGN(A/C)GNGGNGGN(A/T)(C/G)NGA(C/T)(A/T)(A/C)NACNGC-30; (2) primer in the antisense orientation complementary to amino acids 282 to 288 (position 1 to K of KFGG) in AKHSDH (NLSAPGT), with a HindIII site [18]: 50 -GGAAGCTTGTTCC(A/C)GG(A/G)GCAGA(G/T)AGGTTG-30 . Phage plaques eluted in 1 ml SM were screened using PCR (5 l as template) as above with 10 extra min of denaturation at 94 C for capsid disruption prior to the normal cycling. DNA manipulations Recombinant techniques were used for cloning, screening and hybridization [30]. Probes were obtained
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236 by end-labelling PCR products with -32 P-ATP (7000 Ci/mmol, ICN) as described [1, 30]. DNA sequence analysis Sequencing reactions were performed on denatured plasmid DNA using the dideoxy chain-termination method [31] with the Sequenase version 2.0 sequencing kit (Amersham). PR , T7 and internal gene-specific primers were used to anneal to their target sequence. PCR fragments were purified using the Quiaquick kit (Quiagen) prior to sequencing following the same protocol. Contiguous sequences were assembled and analysed using GeneCompar (M. Vauterin, Kortijk). Alignments of nucleotide and amino acid sequences were carried out using the same program. DNA sequences were used to search GenBank and the EMBL Data Library using the BLAST network service. Isolation and analysis of DNA Genomic DNA was isolated from leaves of in vitro grown plantlets as described [12]. The DNA was digested with chosen restriction enzymes, separated by electrophoresis through a 0.8% agarose gel and blotted onto Hybond-N nylon membranes (Amersham). Prehybridization (overnight) and hybridization (two days) were carried out in an oven at 42 C in the following buffer: 5 SSC, 30% or 50% formamide, 10% dextran sulfate, 1% SDS, 2 g/ml sonicated salmon sperm DNA, 10 Denhardt’s solution. Probes were radiolabelled by random oligonucleotide-primed synthesis using the Boehringer Mannheim kit.
Results PCR amplification of a fragment of a monofunctional aspartate kinase (ak-lys) gene PCR amplifications were initially performed on A. thaliana race Columbia genomic DNA using several sets of primers, in particular degenerate primers derived from conserved bacterial lysine-sensitive AK sequences and primers identical to amino acid stretches of the A. thaliana bifunctional AK-HSDH. With the aim to avoid reisolating the gene encoding the bifunctional enzyme, PCR products were blotted and probed with the corresponding ak-hsdh cDNA. Since no (or very low) cross-hybridization was foreseen, amplification fragments approximating the expected size were
directly sequenced and aligned with known bacterial AK-lysine sequences. The 50 -end sequence from a 500 bp fragment, amplified using a degenerate primer (50 ) and a primer specific to the ak-hsdh sequence (30 ) (Figure 1), showed the highest deduced amino acid homology with a region within the E. coli AKIII sequence. The presence of an intron at the 50 end of this fragment was localized by the sudden drop of homology and the typical AG 30 intron consensus end (Figure 5a) [24]. This PCR product was further used to screen both A. thaliana genomic and cDNA phage banks. Isolation of an ak-lys cDNA and two ak-lys genes About 100 000 clones from a lambda UNIZAP II cDNA library were screened using the 500 bp PCR fragment previously end-labelled. Putative positive plaques were pooled per Petri dish for elution. A PCR screening using the same primers as for the probe amplification was done to detect the presence of an ak-lys clone. Only one positive amplification band was detected out of the 10 tubes. Purification of this clone was done by subsequent rounds of radioactive probing and PCR screening. The same procedure was used to screen about 100 000 clones from the genomic lambda FIX bank. After PCR amplification, three tubes presented an amplification product. Two of these had the same molecular weight (around 500 bp) and one was slightly smaller (around 440 bp). To our surprise, all three hybridized to the PCR radioactive probe (Figure 2). This was a first indication for the presence of a second ak-lys gene in A. thaliana. Sequence of the A. thaliana ak-lys cDNA The plasmid cDNA clone in pBluescript SK, was rescued from the UNIZAPII clones using a singlestranded helper phage. Partial sequencing of the ends for one clone revealed sequence similarity with the COOH end of microbial AK-Lys sequences. Figure 1 shows the complete nucleotide sequence and the deduced amino acid sequence of the cDNA insert. Since no in-frame initiation of translation codon could be found, it was supposed that the cDNA was not fulllength. Partial sequencing of the corresponding region in the genomic clone showed this was most likely the case. Thus, the 5 first amino acids (MAATR) and the 50untranslated region are missing in the original cDNA. The corresponding ORF encodes a polypeptide of 569
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Figure 3. Sequence alignment between the two plant monofunctional AKs (AK-LYS1 and cArab-AK-Lys), Escherichia coli AKIII [8], Bacillus subtilis AKII [10], Bacillus sp. AKII [32], and Corynebacterium glutamicum AK [22].
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238 amino acids residues. The NH2 and of AK-Lys1, a chloroplast-localized enzyme [17, 34], is expected to begin with a transit peptide (TP). This TP most likely ends before the typical KFGG motif, a highly conserved amino acid stretch at the NH2 extremity of all AKs identified to date. It would thus be about 90 amino acids long, which is comparable to the length of the AK-HSDH TP [18]. However, the deduced size of the mature AK-Lys1 protein (with +1 on K of KFGG, 479 amino acids (aa)) is considerably smaller than that of the mature AK-HSDH (824 aa). Actually, the AK-Lys1 protein lacks the HSDH domain at the COOH side, thus presenting only two functional regions instead of three: AK (1 to 243 aa) and intermediate domain (ID, 244 to 479 aa), but not HSDH. This is evidence for the monofunctionality of the enzyme, which is comparable with the situation in E. coli. The DPR sequence (Figures 1 and 3) which is highly conserved among all AK domains, and thought to determine kinase activity, is not fully conserved in this AK region: only DP are still present, and R is replaced by T. The major typical monofunctional AK box (TTLGRGGSD) which was chosen to design the successful degenerate primer, is perfectly conserved in the plant sequence. Codon usage of the ak-lys1 gene presents a certain bias, with 60% A or T in the wobble position. This is in agreement with the observed distribution of codon frequency in dicot genes [7]. Comparison of the plant deduced AK-Lys1 amino acid sequence with that of bacterial and yeast monofunctional AKs, and the A. thaliana bifunctional AKHSDH sequence (Figure 4a and b) clearly reveals a higher identity score (37%) between AK-Lys1 and AKIII from E. coli. Only 25% amino acid identity is observed between the plant monofunctional and bifunctional AK and ID regions. The cDNA presents 184 bp 30 -untranslated region in which no perfect consensus polyadenylation signal (AATAAA) could be identified [21]. Identification by partial sequencing of two A. thaliana ak-lys genes Before purifying the three genomic candidates, direct sequencing of one end of the amplified bands was performed to confirm the identity of the clones. Two sequences proved to be identical to the AK-Lys1 cDNA but the last sequence differed to a certain extent. The major variation was due to the presence of an intron at the same position, but differing in size and sequence. A clear intron 30 splice junction consensus end was
Figure 4. The Arabidopsis thaliana AK-Lys1 is most closely related to the E. coli AKIII, after the non-allelic A. thaliana cArab-AK-lys. a (top). A table illustrating the percentage identity and similarity (in parenthesis) at amino acid level between the A. thaliana AKLys1 and its nonallelic cArab-AK-Lys, Escherichia coli AKIII [8], Bacillus subtilis AKII [10], Bacillus species AKII [32], Corynebacterium glutamicum AK [22], Saccharomyces cerevisiae AK [27] and Arabidopsis thaliana AK-HSDH [18]. b (bottom). Dendrogram following UPGMA clustering of the monofunctional AK sequences mentioned in a.
identified [5], beyond which the two deduced amino acid sequences presented high homology (Figure 5). Identity scores of this second sequence with the corresponding region in other available AK sequences were considerably lower than with the plant AK-Lys1 sequence. Interestingly, the DPR motif in this sequence was not fully conserved either: DP was present but R was replaced by N. These results led to the conclusion that another ak gene is present in A. thaliana, and considering that it was identified with primers used to clone ak-lys1 with which it shares highest identity percentage, it is most likely to be monofunctional too. This was further confirmed by the results presented in accompanying paper by Tang et al. [33]. Amino acid identity between both complete sequences is of ca. 69%, and similarity percentage reaches 79%, which show the genes are rather divergent (Figure 4a). The two TPs show practically no identity at amino acid sequence level, which is not expected since typical content (for example, rich in Ser and Thr) rather than consensus
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Figure 5. (a) and (b) Partial nucleotide and deduced amino acid sequences from the two genomic clones. The 30 AG consensus end of the intron is underlined, small caps are used to indicate intron sequence. (c) Amino acid alignment between the two deduced partial sequences.
sequences characterize these targeting peptides (Figure 3). Restriction analysis and partial sequencing of the three genomic clones indicate that two are identical. Preliminary gene structure analysis revealed a high number of short introns interrupting the two coding sequences. Is there a small ak-lys gene family in A. thaliana? To assess the number of ak-lys genes in the A. thaliana genome, DNA was digested with three different restriction enzymes and hybridized with an ak-lys1 EcoRV fragment. The simple banding pattern observed for all restrictions in stringent hybridization conditions, tends to imply the presence of a single ak-lys1 gene per haploid genome (Figure 6). Other probes, shorter and centered around the major typical ak-lys box (TTLGRGGSD), reveal the presence of the second gene identified (Figure 7). The presence of at least two genes in A. thaliana coding for a monofunctional AK is thus also confirmed by Southern analysis.
Discussion This report describes the isolation of a cDNA from A. thaliana that encodes a monofunctional AK protein. Two lines of evidence to sustain this are (1) the higher amino acid identity score between the plant sequence and the E. coli AKIII (31%); (2) the absence of HSDH function at the COOH of the deduced amino acid sequence. There have been no previous reports on the cloning of monofunctional AK-encoding genes in plants. Translation of the ORF reveals the KFGG motif, typical of the NH2 end of all AKs isolated to date, although its function has not yet been determined. Upstream of this amino acid box are ca. 90 amino acids, typical of chloroplast transit peptides (rich in serine, threonine, and small hydrophobic amino acids). This is expected through the biochemical evidence available from the literature, in which AK activity is exclusively associated with the plastidic subcellular fraction [23, 34]. The exact amino-terminus of the mature AK-Lys protein is unknown, just as it is for the bifunctional AK-HSDH. It can however be speculated on the basis of sequence alignments between all AKs available that the KFGG motif itself or at most a couple amino acids
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Figure 6. Southern blot analysis of the ak-lys1 gene in A. thaliana. Equal amounts of genomic DNA (10 g) restricted with BamHI, EcoRI, HindIII and HincII were probed with the full-length ak-lys1 cDNA.
upstream are bordering the mature protein. Another common feature to all AKs characterized so far, and to a few other kinases as well, is the presence of the DPR motif, to which the kinase activity has been related. The two new lysine-sensitive AKs from A. thaliana present only the DP amino acid pair, which questions the integrity of this site. The deduced molecular mass of the protein is about 52.5 kDa, which approaches the 50 kDa determined for the E. coli AKIII subunit. In the latter, the holoenzyme is a homodimer, whereas in Bacillus sp. and Corynebacterium sp. a heterodimer of 47 and 18 kDa has been determined [22, 25]. Lysine-sensitive AKs have been purified to near homogeneity in carrot [28] and in maize [2, 15] but estimations of holoenzyme molecular weight and subunit composition vary greatly. Heterologous expression of the plant ak-lys1 cDNA in E. coli will be attempted to carry out biochemical analyses to uncover this aspect. The presence of a second non-allelic monofunctional AK was detected by PCR product sequencing and by Southern analysis, and confirmed by the results described in the accompanying paper. The predicted A. thaliana proteins are 69% identical to one another, which is not so conserved phylogenetically and indic-
Figure 7. Southern blot analysis of the monofunctional ak genes in A. thaliana. Equal amounts of genomic DNA (10 g) restricted with HindIII plus SacI (lane 1), SalI (lane 2). NcoI plus SalI (lane 3), and probed successively with (a, left) the complete ak-lys1 cDNA and (b, right) a 248 bp PCR fragment including the ‘lysine box’. Both blots were hybridized and washed mildly. Arrows show size markers, triangles indicate extra bands appearing with the PCR probe.
ates that the duplication event is not recent. Two lysinesensitive AKs have been demonstrated by genetic studies in barley [3] and in maize [13, 14]: in both cases, two mutations rendering AK less sensitive to lysinefeedback control were mapped to separate structural loci. The presence of two AKs sensitive to lysine has however never been shown for dicots, either genetically of biochemically. In all cases, this duplication raises the question of the respective role of each gene within the aspartate biosynthetic pathway. Both proteins are targeted to the chloroplast, as most enzymes involved in essential amino acid biosynthesis, but in contrast with enzymes implied in nitrogen assimilation (such as glutamine synthetase). A more detailed study of their regulation in relation to particular cell types (for example, mesophyll versus phloem cells), stages of development (such as meristems versus full-grown
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241 leaf tissues), environmental stimuli (availability of N or light), is underway. Although Southern analyses in Arabidopsis indicates the presence of a single gene for each AK sensitive to lysine and for the bifunctional AK-HSDH, small genes families may be expected in other species as it is the case for the maize AK-HSDH encoded by three non-allelic genes [26].
11.
12. 13.
Acknowledgements Drs Goodman (Boston, USA) and Trezzini (Max Planck Institute, Germany) are thanked for providing the A. thaliana genomic and cDNA banks, respectively. Gad Galili is acknowledged for providing the sequence of the other monofunctional AK (cArabAK-Lys) prior to publication. V.F. is a recipient of a post-doctoral fellowship of the ‘Nationaal Fonds voor Wetenschappelijk Onderzoek (NFWO)’. This project and M.V. are supported by the ‘Geconcerteerde Onderzoeksactie’ 92-97-134 and the NATO Collaborative Research Grant Programme CRG 900601.
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17. 18.
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