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Isolation and characterisation of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein
Plant Science 162 (2002) 233– 238 www.elsevier.com/locate/plantsci
Isolation and characterisation of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein Marı´a-Cruz Gonza´lez a, Cristina Echevarrı´a b, Jean Vidal c, Francisco J. Cejudo a,* a
Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Centro de In6estigaciones Cientı´ficas ‘Isla de la Cartuja’, A6da Ame´rico Vespucio s/n, 41092 Se6illa, Spain b Departamento de Biologı´a Vegetal, Facultad de Biologı´a, Uni6ersidad de Se6illa, Se6illa, Spain c Institut de Biotechnologie des Plantes, Centre d’Orsay, Uni6ersite´ de Paris-Sud, UMR CNRS 8618, Baˆtiment 630, 91405 Orsay Cedex, France Received 29 May 2001; received in revised form 5 September 2001; accepted 5 October 2001
Abstract A phosphoenolpyruvate carboxylase gene (referred to as Ppc1 ) was isolated from a wheat genomic library and sequenced in both strands. The deduced protein sequence is encoded by 10 exons and shows extensive similarities to that of other plant PEPCs sequenced so far, with respect to the predicted motives involved in the structure (b-strands and a-helices) and function (active site, inhibitor site, N- and C-terminus) of the enzyme. Modelling of the 3D-structure of the wheat PEPC monomer using the Swiss-Model programme and the Escherichia coli PEPC monomer 3D-structure as a reference revealed that these motives and corresponding key residues share very similar positions in the folded plant and bacterial polypeptides. Interestingly, the 5%-flanking sequence of Ppc1 gene contains various putative regulatory cis-elements including an ABRE box. Relative RT-PCR analysis showed a ubiquitous expression of Ppc1 gene in developing and germinating wheat seeds as well as in developing seedlings. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phosphoenolpyruvate carboxylase; Gene; Modelling; Triticum aesti6um
1. Introduction Phosphoenolpyruvate carboxylase (PEPC, E.C. 4.1.1.31) is a homotetrameric enzyme that catalyses the b-carboxylation of PEP by HCO− 3 in the presence of a divalent cation to yield Pi and oxaloacetate. This cytosolic enzyme is widely distributed in most plant tissues, green algae and microorganisms but not in animal cells [1]. In plants, PEPC fulfils various physiological roles e.g. the well-documented photosynthetic CO2 fixation in C4 and CAM plants, and the anaplerotic pathway [1 – 3]. PEPC is subject to allosteric regulation by opposing metabolite effectors, malate (negative feedback) and glucose-6P (positive). The metabolite regulation of PEPC is modulated by reversible phosphorylation of a regulatory serine in the plant invariant domain of the * Corresponding author. Tel.: + 34-954-48-9511; fax: +34-954-460065. E-mail address: [email protected] (F.J. Cejudo).
enzyme’s N-terminus [1,3,4]. In C4 plants, this regulatory mechanism involves a dedicated, Ca2 + -independent protein kinase, which depends on a complex light-transduction cascade [5]. PEPC phosphorylation by the requisite protein kinase was also found to occur for the non-photosynthetic PEPCs in C3 plants (reviewed in [1,3]). Although a relatively large number of PEPC cDNA sequences have been reported, PEPC genes and promoter sequences remain poorly documented [1]. However, the availability of these promoters may be of great help to understand the regulation of PEPC gene expression in transgenic plants [1,5]. In this paper, we report on the isolation and characterisation of one member of the wheat PEPC gene family, which is expressed in all organs and developmental stages analysed. The encoded protein is compared with those from other plant and bacterial (Escherichia coli ) PEPCs, including modelling of the 3D-structure.
0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 4 8 - 9
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Fig. 1. Ppc1 gene structure. The nucleotide sequence is presented in lower case. The single letter amino acid code above the first base of each codon is used to show the open reading frames in the exons of the gene. Putative TATA box, intron/exon junctions, and polyadenylation signals are underlined. *, Stop codon. Box 1, ABA-responsive element (ABRE). Arrows underline oligonucleotides Ppc1 -A and Ppc1 -B. The nucleotide sequence data reported in this paper appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ007705.
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and smaller, overlapping fragments subcloned in pBluescript KS vector and sequenced in both strands.
2.3. RNA isolation and relati6e RT-PCR analysis
Fig. 2. Deduced amino acid sequence of the wheat PEPC. The N-terminal phosphorylation domain containing the regulatory Ser18 and the motifs forming the active and inhibitor sites are shown in italic, with underlined key residues. The second Arg in the glycinerich loop F H6 G R6 G G T V G R6 G G is shared by the active and inhibitor sites.
2. Methods
2.1. Plant material Wheat (Triticum aesti6um, cv Chinese Spring) plants were cultivated under controlled-environment conditions in a greenhouse under a 16-h day/8-h night cycle at 22–25 °C. Seeds were harvested at different stages of development, frozen in liquid nitrogen and kept at − 80 °C until required. Mature seeds were sterilised in 2% (v/v) NaOCl for 20 min and washed twice with sterile water, once with 0.1 M HCl, and then thoroughly with sterile water. Seeds were allowed to germinate at room temperature on filter paper soaked with water for periods of up to 4 days.
2.2. Wheat PEPC gene cloning A wheat genomic library constructed in lFIX (Stratagene) was screened with 32P-labelled wheat partial cDNA encoding the C-terminus of the protein [6]. The library (ca. 800,000 pfu) was plated, transferred to Hybond-N filters (Amersham, UK) and hybridised at 60 °C according to manufacturer’s instructions. Three positive plaques were obtained, purified and their DNA inserts analysed by restriction enzyme analysis and hybridisation. A 7.4 kb SalI fragment containing the complete sequence of wheat PEPC was cloned in pBluescript KS, a complete restriction map was established
Total RNA was isolated as previously reported [6] from dissected aleurone layers and scutellum of germinating wheat seeds, shoots and roots from developing seedlings, and developing seeds harvested at 5, 8 and 30 days after pollination (dap). To analyse the presence of Ppc1 mRNA, two oligonucleotides were designed Ppc1 A (5%-GCTGGAGGATACCCTCATCTT) and Ppc1 -B (5%-GGCAGATGCAGCAACATCCGC) in order to amplify a 171-bp PCR product from the 3%-untranslated sequence of Ppc1 mRNA. For relative RT-PCR analysis, total RNA (0.5 mg) was treated with 2 U of DNase I (Ambion) for 1 h at 37 °C, then DNase I was inactivated by treatment with DNase I Inactivating Reagent (Ambion). This DNA-free RNA was retrotranscribed with random primers and SuperScript retrotranscriptase (Gibco-BRL), at 42 °C for 1 h. Samples of the first-strand cDNA were then used in PCR reactions using Ppc1 -A and Ppc1 -B oligonucleotides and 18S rRNA primers and competimers (QuantumRNA 18S Internal Standards, Ambion) according to manufacturers instructions, as internal controls of the amount and quality of RNA. Control reactions were performed using as template non-retrotranscribed RNA to rule out possible amplification from contaminating genomic DNA.
3. Results and discussion
3.1. Isolation and characterisation of a genomic clone encoding wheat PEPC A 7.4 kb SalI fragment was isolated which contained a complete wheat PEPC gene. The nucleotide sequence, location of introns (deduced from comparison with the cDNA sequence), and deduced polypetide are shown in Fig. 1. Gene structure of the wheat PEPC gene, referred to hereafter as Ppc1, is composed of 10 exons. The open reading frame encodes a deduced polypeptide of 972 amino acids with an expected molecular mass of 110.1 kDa. The intron/exon junctions are as expected for plant genes and are underlined in Fig. 1. However, the sequence around the translation start codon of Ppc1 is more closely related to that found in genes from animal cells [7,8]. A putative TATA box is present upstream the translational start codon, and putative polyadenylation signals (underlined in Fig. 1) are found downstream the stop codon. A search of putative cis-elements in the 5%-flanking sequence of Ppc1 gene showed the presence of an ABRE (ABA-responsive element) (Box 1 in Fig. 1). This element is found in the promoter
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of genes involved in the response to several environmental stresses [9], thus suggesting the possibility that PEPC is involved in the response of wheat to abiotic stress. The deduced polypeptide sequence displayed a high degree of identity (75–93%) to other plant PEPCs, but it was found to diverge significantly from prokaryotic PEPCs (32 –43%). However, comparing the wheat PEPC sequence to that of the recently crystallised E. coli PEPC revealed that most of the essential domains and amino acid residues involved in PEPC structure, activity and regulation are shared by both polypeptides [10]. Contributing to the probable active site are: His179 (TAHPT); Lys607 (GYSDSxKDAG); Arg457 (DxRQES); His640, Arg642 and Arg648 (in the Glyrich loop FHGRGGxxGRGG), and Arg760 (GSRPxKRxP) (Fig. 2). Residues of the inhibitor (malate) site, which likely correspond to the aspartate binding site in the bacterial PEPC, are Arg648 (in the Gly-rich loop FHGRGGxxGRGG), Lys836 (EMVFAK), Arg895 (LRxxY) and Asn970 (NTG) (Fig. 2).
An important property of plant PEPCs is its regulation by reversible phosphorylation, which occurs on a Ser residue at the enzyme’s N-terminus (reviewed in [1,3,4]). In wheat PEPC, this Ser residue is at position 18 in the plant invariant phosphorylation domain Acid–BaseXXSIDAQLR (Fig. 2). Finally, seven highly conserved Cys residues (positions 198, 310, 337, 420, 425, 427, 688) may be involved in redox regulation of PEPC activity [11,12]. The crystal structure of the E. coli PEPC has been recently determined by X-ray diffraction methods using an enzyme complexed with the allosteric inhibitor L-aspartate [10]. To clarify the spatial organisation of the conserved domains in the wheat PEPC monomer, we modelled its 3D-structure using the Swiss-Model programme [13–15] and the prokaryotic monomer as a reference. The global shape of the folded polypeptides was found to be highly similar (Fig. 3A), with the notable exception of additional b-strands in the plant PEPC subunit. Common structural features are: the central b-barrel organising numerous a-helices and loops to form the active and inhibitor sites (the corre-
Fig. 3. Ribbon drawings of the wheat and E. coli PEPC monomer (A). Modelling was performed using the Swiss-Model programme. The central b-barrel is shown in yellow, the a-helices in red and the connecting loops in white. The additional b-strands (in yellow) are found on the right side of the wheat PEPC monomer. (B) 3D representation of the active (blue) and inhibitor (red) site; the key residues are numbered: 1, Arg457; 2, Arg760; 3, His179; 4, Lys607; 5, Arg642; 6, His640; 7, Arg648; 8, Asn970; 9, Lys836; 10, Arg895.
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Fig. 4. Phylogenetic tree for plant PEPCs. Dengrogram was constructed by multiple alignment of plant PEPC amino acid sequences obtained from SWISS PROT database. Distances between sequences was obtained with algorithm DISTANCES and the method ‘Uncorrected Distances’. Then algorithm GROWTREE and the method ‘Neighbor-joining’ were used to construct the dendrogram.
sponding important residues are distributed in an almost identical manner, as shown in Fig. 3B), the burried, C-terminal hydrophobic a-helice with terminal, non-helical xNTG containing Asn970 (which is part of
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the inhibitor site). The C-terminus sequence of the enzyme subunit seems to be important for stabilising its tetrameric structure in both plant and prokaryotic PEPCs [10,16]. In the E. coli enzyme, it has been proposed that the loop FHGRGGxxGRGGxP (Hys640, Arg642 and Arg648) contributes to catching substrate molecules at the active site and forms a lid to protect the reaction intermediates from attack by surrounding water [10]. Also, Arg648 is shared by both the active and inhibitor sites. Removal of this residue from the active site upon binding of the effector (aspartate/malate) perturbs substrate binding and, thus, accounts for metabolite-induced loss of catalytic activity. The position of this residue in Fig. 3B (E. coli ) corresponds to the constrained (inhibited) structure of the bacterial enzyme, which was crystallised in the presence of L-aspartate. Interestingly, the modelled plant enzyme also shows displacement of the corresponding Arg648 (Fig. 3B, wheat). Finally, the bacterial subunit does not possess the N-terminus domain, which, is not included in the plant subunit. PEPC is considered as an excellent molecular marker for plant phylogenetic studies [5,17–19]. To establish the phylogenetic position of Ppc1, a dendrogram was constructed using sequences of the C-terminus region (residues 603–972 of the wheat enzyme, Fig. 1) of plant PEPCs available in databases. As mentioned above, this PEPC region includes highly conserved domains important for protein structure and function. No prokaryotic PEPC sequences were included in this study since they form a different group according to the classification established by Toh et al. [18]. In this dendrogram Ppc1 is grouped with C3-type PEPCs from the monocots Sorghum 6ulgare and Zea mays in a branch which also includes a CAM-type PEPC from M. crystallinum (Fig. 4).
Fig. 5. Relative RT-PCR analysis of Ppc1 gene expression in wheat seeds and seedlings. Total RNA was isolated from developing wheat seeds harvested at the indicated days after pollination (A), from aleurone layers and scutellum dissected from seeds after 1 – 4 days of imbibition (B), and from roots or shoots from 2- to 4-day-old developing seedlings (C). After retrotranscription and RT-PCR in the presence of Ppc1 specific primers (Ppc1 -A and Ppc1 -B) and 18S rRNA primers and competimers, the PCR products were fractionated on agarose gels.
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3.2. Relati6e RT-PCR analysis of Ppc1 expression
References
In a previous report [6] we described PEPC expression in wheat seeds and seedlings based on northernblot analysis using as probe a cDNA expanding the C-terminal region (331 amino acids) of wheat Ppc1 gene. A single band was detected, which probably corresponded to transcripts from the different members of the PEPC gene family of wheat. To address the pattern of expression of Ppc1 gene, specific primers were designed, described in Section 2, to amplify a 171-bp fragment of the 3%-untranslated sequence of Ppc1 mRNA in Relative RT-PCR experiments (Fig. 5). This sensitive technique showed the presence of Ppc1 transcripts in any wheat organ or developmental stage analysed: developing seeds (Fig. 5A), aleurone and scutellum from germinating seeds (Fig. 5B), and roots and shoots from developing seedlings (Fig. 5C). Thus showing that Ppc1 gene is a ubiquitously expressed member of the wheat PEPC gene family. In spite of the wide expression of Ppc1 gene, it should be noted that during seed development a high accumulation of Ppc1 transcripts was detected at early stages (5 days) that progressively decreased to be almost undetectable at later stages of seed development (Fig. 5A). The western-blot analysis of PEPC polypeptides during wheat seed development showed the presence of an invariable 103- and a 108-kDa subunit, which was abundant at early stages of development and progressively disappeared at later stages [6], a pattern which matches the expression of Ppc1 gene (Fig. 5A). Since the PEPC polypeptide deduced from Ppc1 gene shows an expected molecular mass of 110 kDa, which fits reasonably well with the 108 kDa estimated for this PEPC subunit based on SDS-PAGE, it is likely that Ppc1 gene encodes the higher molecular weight PEPC polypeptide of wheat.
[1] R. Chollet, J. Vidal, M.H. O’leary, Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants, Annu. Rev. Plant Physiol. Mol. Biol. 47 (1996) 273 – 298. [2] H.C. Huppe, D.H. Turpin, Integration of carbon and nitrogen metabolism in plant and algal cells, Annu. Rev. Plant Physiol. Mol. Biol. 45 (1994) 577 – 607. [3] J. Vidal, R. Chollet, Regulatory phosphorylation of C4 PEP carboxylase, Trends Plant Sci. 2 (1997) 203 – 241. [4] H.G. Nimmo, The regulation of phosphoenolpyruvate carboxylase in CAM plants, Trends Plant Sci. 5 (2000) 75 – 79. [5] L. Lepiniec, J. Vidal, R. Chollet, P. Gadal, C. Cre´ tin, Phosphoenolpyruvate carboxylase: structure, regulation and evolution, Plant Sci. 99 (1994) 111 – 124. [6] M.C. Gonza´ lez, L. Osuna, C. Echevarria, J. Vidal, F.J. Cejudo, Expression and localization of phosphoenolpyruvate carboxylase in developing and germinating wheat grains, Plant Physiol. 116 (1998) 1249 – 1258. [7] M. Kozak, Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNA, Nucl. Acids Res. 12 (1984) 857 – 872. [8] H.A. Lutcke, K.C. Chow, F.S. Mickel, K.A. Moss, H.F. Kern, G.A. Scheele, Selection of AUG initiation codons differs in plants and animals, EMBO J. 6 (1987) 43 – 48. [9] P.K. Busk, M. Page`s, Regulation of abscisic acid-induced transcription, Plant Mol. Biol. 37 (1998) 425 – 435. [10] Y. Kai, H. Matsumura, T. Inoue, K. Terada, Y. Nagara, T. Yoshinaga, A. Kihara, K. Tsumura, K. Izui, Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition, Proc. Natl. Acad. Sci. USA 96 (1999) 823 – 828. [11] A.A. Iglesias, C.S. Andreo, On the molecular mechanism of maize PEPC activation by thiol compounds, Plant Physiol. 75 (1984) 983 – 987. [12] T.P. Chardot, R.T. Wedding, Role of cysteine in activation and allosteric regulation of maize PEPC, Plant Physiol. 98 (1992) 780 – 783. [13] M.C. Peitsch, Protein modelling by E-mail, Bio/Technology 13 (1995) 658 – 660. [14] M.C. Peitsch, ProMod and Swiss-Model: internet-based tools for automated comparative protein modelling, Biochem. Soc. Trans. 24 (1996) 274 – 279. [15] N. Guex, M.C. Peitsch, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modelling, Electrophoresis 18 (1997) 2714 – 2723. [16] L. Dong, S. Patil, S.A. Condon, E.J. Haas, R. Chollet, The conserved C-terminal tetrapeptide of sorghum (C4) phosphoenolpyruvate carboxylase is indispensable for maximal catalytic activity but not for homotetramer formation, Arch. Biochem. Biophys. 371 (1999) 124 – 128. [17] W. Poetsch, J. Hermans, P. Westhoff, Multiple cDNAs of phosphoenolpyruvate carboxylase in the C4 dicot Fla6eria triner6ia, FEBS Lett. 292 (1991) 133 – 136. [18] H. Toh, T. Kawamura, K. Izui, Molecular evolution of phosphoenolpyruvate carboxylase, Plant Cell Environ. 17 (1994) 31 –43. [19] H.H. Gehrig, V. Heute, M. Kluge, Toward a better knowledge of the molecular evolution of phosphoenolpyruvate carboxylase by comparison of partial cDNA sequences, J. Mol. Evol. 46 (1998) 107 – 114.
Acknowledgements Thanks are due to Dr C. Hartman for generous gift of the wheat genomic library and to Dr Michael Hodges for in silico modelling of PEPC and helpful discussion of the manuscript. This work was supported by grant PB97-0745 from Direccio´ n General de Ensen˜ anza Superior, Ministerio de Educacio´ n y Cultura and Grant CVI 182 from Junta de Andalucı´a (Spain).
Isolation and characterisation of a wheat phosphoenolpyruvate carboxylase gene. Modelling of the encoded protein Marı´a-Cruz Gonza´lez a, Cristina Echevarrı´a b, Jean Vidal c, Francisco J. Cejudo a,* a
Instituto de Bioquı´mica Vegetal y Fotosı´ntesis, Centro de In6estigaciones Cientı´ficas ‘Isla de la Cartuja’, A6da Ame´rico Vespucio s/n, 41092 Se6illa, Spain b Departamento de Biologı´a Vegetal, Facultad de Biologı´a, Uni6ersidad de Se6illa, Se6illa, Spain c Institut de Biotechnologie des Plantes, Centre d’Orsay, Uni6ersite´ de Paris-Sud, UMR CNRS 8618, Baˆtiment 630, 91405 Orsay Cedex, France Received 29 May 2001; received in revised form 5 September 2001; accepted 5 October 2001
Abstract A phosphoenolpyruvate carboxylase gene (referred to as Ppc1 ) was isolated from a wheat genomic library and sequenced in both strands. The deduced protein sequence is encoded by 10 exons and shows extensive similarities to that of other plant PEPCs sequenced so far, with respect to the predicted motives involved in the structure (b-strands and a-helices) and function (active site, inhibitor site, N- and C-terminus) of the enzyme. Modelling of the 3D-structure of the wheat PEPC monomer using the Swiss-Model programme and the Escherichia coli PEPC monomer 3D-structure as a reference revealed that these motives and corresponding key residues share very similar positions in the folded plant and bacterial polypeptides. Interestingly, the 5%-flanking sequence of Ppc1 gene contains various putative regulatory cis-elements including an ABRE box. Relative RT-PCR analysis showed a ubiquitous expression of Ppc1 gene in developing and germinating wheat seeds as well as in developing seedlings. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Phosphoenolpyruvate carboxylase; Gene; Modelling; Triticum aesti6um
1. Introduction Phosphoenolpyruvate carboxylase (PEPC, E.C. 4.1.1.31) is a homotetrameric enzyme that catalyses the b-carboxylation of PEP by HCO− 3 in the presence of a divalent cation to yield Pi and oxaloacetate. This cytosolic enzyme is widely distributed in most plant tissues, green algae and microorganisms but not in animal cells [1]. In plants, PEPC fulfils various physiological roles e.g. the well-documented photosynthetic CO2 fixation in C4 and CAM plants, and the anaplerotic pathway [1 – 3]. PEPC is subject to allosteric regulation by opposing metabolite effectors, malate (negative feedback) and glucose-6P (positive). The metabolite regulation of PEPC is modulated by reversible phosphorylation of a regulatory serine in the plant invariant domain of the * Corresponding author. Tel.: + 34-954-48-9511; fax: +34-954-460065. E-mail address: [email protected] (F.J. Cejudo).
enzyme’s N-terminus [1,3,4]. In C4 plants, this regulatory mechanism involves a dedicated, Ca2 + -independent protein kinase, which depends on a complex light-transduction cascade [5]. PEPC phosphorylation by the requisite protein kinase was also found to occur for the non-photosynthetic PEPCs in C3 plants (reviewed in [1,3]). Although a relatively large number of PEPC cDNA sequences have been reported, PEPC genes and promoter sequences remain poorly documented [1]. However, the availability of these promoters may be of great help to understand the regulation of PEPC gene expression in transgenic plants [1,5]. In this paper, we report on the isolation and characterisation of one member of the wheat PEPC gene family, which is expressed in all organs and developmental stages analysed. The encoded protein is compared with those from other plant and bacterial (Escherichia coli ) PEPCs, including modelling of the 3D-structure.
0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 ( 0 1 ) 0 0 5 4 8 - 9
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Fig. 1. Ppc1 gene structure. The nucleotide sequence is presented in lower case. The single letter amino acid code above the first base of each codon is used to show the open reading frames in the exons of the gene. Putative TATA box, intron/exon junctions, and polyadenylation signals are underlined. *, Stop codon. Box 1, ABA-responsive element (ABRE). Arrows underline oligonucleotides Ppc1 -A and Ppc1 -B. The nucleotide sequence data reported in this paper appears in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number AJ007705.
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and smaller, overlapping fragments subcloned in pBluescript KS vector and sequenced in both strands.
2.3. RNA isolation and relati6e RT-PCR analysis
Fig. 2. Deduced amino acid sequence of the wheat PEPC. The N-terminal phosphorylation domain containing the regulatory Ser18 and the motifs forming the active and inhibitor sites are shown in italic, with underlined key residues. The second Arg in the glycinerich loop F H6 G R6 G G T V G R6 G G is shared by the active and inhibitor sites.
2. Methods
2.1. Plant material Wheat (Triticum aesti6um, cv Chinese Spring) plants were cultivated under controlled-environment conditions in a greenhouse under a 16-h day/8-h night cycle at 22–25 °C. Seeds were harvested at different stages of development, frozen in liquid nitrogen and kept at − 80 °C until required. Mature seeds were sterilised in 2% (v/v) NaOCl for 20 min and washed twice with sterile water, once with 0.1 M HCl, and then thoroughly with sterile water. Seeds were allowed to germinate at room temperature on filter paper soaked with water for periods of up to 4 days.
2.2. Wheat PEPC gene cloning A wheat genomic library constructed in lFIX (Stratagene) was screened with 32P-labelled wheat partial cDNA encoding the C-terminus of the protein [6]. The library (ca. 800,000 pfu) was plated, transferred to Hybond-N filters (Amersham, UK) and hybridised at 60 °C according to manufacturer’s instructions. Three positive plaques were obtained, purified and their DNA inserts analysed by restriction enzyme analysis and hybridisation. A 7.4 kb SalI fragment containing the complete sequence of wheat PEPC was cloned in pBluescript KS, a complete restriction map was established
Total RNA was isolated as previously reported [6] from dissected aleurone layers and scutellum of germinating wheat seeds, shoots and roots from developing seedlings, and developing seeds harvested at 5, 8 and 30 days after pollination (dap). To analyse the presence of Ppc1 mRNA, two oligonucleotides were designed Ppc1 A (5%-GCTGGAGGATACCCTCATCTT) and Ppc1 -B (5%-GGCAGATGCAGCAACATCCGC) in order to amplify a 171-bp PCR product from the 3%-untranslated sequence of Ppc1 mRNA. For relative RT-PCR analysis, total RNA (0.5 mg) was treated with 2 U of DNase I (Ambion) for 1 h at 37 °C, then DNase I was inactivated by treatment with DNase I Inactivating Reagent (Ambion). This DNA-free RNA was retrotranscribed with random primers and SuperScript retrotranscriptase (Gibco-BRL), at 42 °C for 1 h. Samples of the first-strand cDNA were then used in PCR reactions using Ppc1 -A and Ppc1 -B oligonucleotides and 18S rRNA primers and competimers (QuantumRNA 18S Internal Standards, Ambion) according to manufacturers instructions, as internal controls of the amount and quality of RNA. Control reactions were performed using as template non-retrotranscribed RNA to rule out possible amplification from contaminating genomic DNA.
3. Results and discussion
3.1. Isolation and characterisation of a genomic clone encoding wheat PEPC A 7.4 kb SalI fragment was isolated which contained a complete wheat PEPC gene. The nucleotide sequence, location of introns (deduced from comparison with the cDNA sequence), and deduced polypetide are shown in Fig. 1. Gene structure of the wheat PEPC gene, referred to hereafter as Ppc1, is composed of 10 exons. The open reading frame encodes a deduced polypeptide of 972 amino acids with an expected molecular mass of 110.1 kDa. The intron/exon junctions are as expected for plant genes and are underlined in Fig. 1. However, the sequence around the translation start codon of Ppc1 is more closely related to that found in genes from animal cells [7,8]. A putative TATA box is present upstream the translational start codon, and putative polyadenylation signals (underlined in Fig. 1) are found downstream the stop codon. A search of putative cis-elements in the 5%-flanking sequence of Ppc1 gene showed the presence of an ABRE (ABA-responsive element) (Box 1 in Fig. 1). This element is found in the promoter
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of genes involved in the response to several environmental stresses [9], thus suggesting the possibility that PEPC is involved in the response of wheat to abiotic stress. The deduced polypeptide sequence displayed a high degree of identity (75–93%) to other plant PEPCs, but it was found to diverge significantly from prokaryotic PEPCs (32 –43%). However, comparing the wheat PEPC sequence to that of the recently crystallised E. coli PEPC revealed that most of the essential domains and amino acid residues involved in PEPC structure, activity and regulation are shared by both polypeptides [10]. Contributing to the probable active site are: His179 (TAHPT); Lys607 (GYSDSxKDAG); Arg457 (DxRQES); His640, Arg642 and Arg648 (in the Glyrich loop FHGRGGxxGRGG), and Arg760 (GSRPxKRxP) (Fig. 2). Residues of the inhibitor (malate) site, which likely correspond to the aspartate binding site in the bacterial PEPC, are Arg648 (in the Gly-rich loop FHGRGGxxGRGG), Lys836 (EMVFAK), Arg895 (LRxxY) and Asn970 (NTG) (Fig. 2).
An important property of plant PEPCs is its regulation by reversible phosphorylation, which occurs on a Ser residue at the enzyme’s N-terminus (reviewed in [1,3,4]). In wheat PEPC, this Ser residue is at position 18 in the plant invariant phosphorylation domain Acid–BaseXXSIDAQLR (Fig. 2). Finally, seven highly conserved Cys residues (positions 198, 310, 337, 420, 425, 427, 688) may be involved in redox regulation of PEPC activity [11,12]. The crystal structure of the E. coli PEPC has been recently determined by X-ray diffraction methods using an enzyme complexed with the allosteric inhibitor L-aspartate [10]. To clarify the spatial organisation of the conserved domains in the wheat PEPC monomer, we modelled its 3D-structure using the Swiss-Model programme [13–15] and the prokaryotic monomer as a reference. The global shape of the folded polypeptides was found to be highly similar (Fig. 3A), with the notable exception of additional b-strands in the plant PEPC subunit. Common structural features are: the central b-barrel organising numerous a-helices and loops to form the active and inhibitor sites (the corre-
Fig. 3. Ribbon drawings of the wheat and E. coli PEPC monomer (A). Modelling was performed using the Swiss-Model programme. The central b-barrel is shown in yellow, the a-helices in red and the connecting loops in white. The additional b-strands (in yellow) are found on the right side of the wheat PEPC monomer. (B) 3D representation of the active (blue) and inhibitor (red) site; the key residues are numbered: 1, Arg457; 2, Arg760; 3, His179; 4, Lys607; 5, Arg642; 6, His640; 7, Arg648; 8, Asn970; 9, Lys836; 10, Arg895.
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Fig. 4. Phylogenetic tree for plant PEPCs. Dengrogram was constructed by multiple alignment of plant PEPC amino acid sequences obtained from SWISS PROT database. Distances between sequences was obtained with algorithm DISTANCES and the method ‘Uncorrected Distances’. Then algorithm GROWTREE and the method ‘Neighbor-joining’ were used to construct the dendrogram.
sponding important residues are distributed in an almost identical manner, as shown in Fig. 3B), the burried, C-terminal hydrophobic a-helice with terminal, non-helical xNTG containing Asn970 (which is part of
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the inhibitor site). The C-terminus sequence of the enzyme subunit seems to be important for stabilising its tetrameric structure in both plant and prokaryotic PEPCs [10,16]. In the E. coli enzyme, it has been proposed that the loop FHGRGGxxGRGGxP (Hys640, Arg642 and Arg648) contributes to catching substrate molecules at the active site and forms a lid to protect the reaction intermediates from attack by surrounding water [10]. Also, Arg648 is shared by both the active and inhibitor sites. Removal of this residue from the active site upon binding of the effector (aspartate/malate) perturbs substrate binding and, thus, accounts for metabolite-induced loss of catalytic activity. The position of this residue in Fig. 3B (E. coli ) corresponds to the constrained (inhibited) structure of the bacterial enzyme, which was crystallised in the presence of L-aspartate. Interestingly, the modelled plant enzyme also shows displacement of the corresponding Arg648 (Fig. 3B, wheat). Finally, the bacterial subunit does not possess the N-terminus domain, which, is not included in the plant subunit. PEPC is considered as an excellent molecular marker for plant phylogenetic studies [5,17–19]. To establish the phylogenetic position of Ppc1, a dendrogram was constructed using sequences of the C-terminus region (residues 603–972 of the wheat enzyme, Fig. 1) of plant PEPCs available in databases. As mentioned above, this PEPC region includes highly conserved domains important for protein structure and function. No prokaryotic PEPC sequences were included in this study since they form a different group according to the classification established by Toh et al. [18]. In this dendrogram Ppc1 is grouped with C3-type PEPCs from the monocots Sorghum 6ulgare and Zea mays in a branch which also includes a CAM-type PEPC from M. crystallinum (Fig. 4).
Fig. 5. Relative RT-PCR analysis of Ppc1 gene expression in wheat seeds and seedlings. Total RNA was isolated from developing wheat seeds harvested at the indicated days after pollination (A), from aleurone layers and scutellum dissected from seeds after 1 – 4 days of imbibition (B), and from roots or shoots from 2- to 4-day-old developing seedlings (C). After retrotranscription and RT-PCR in the presence of Ppc1 specific primers (Ppc1 -A and Ppc1 -B) and 18S rRNA primers and competimers, the PCR products were fractionated on agarose gels.
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3.2. Relati6e RT-PCR analysis of Ppc1 expression
References
In a previous report [6] we described PEPC expression in wheat seeds and seedlings based on northernblot analysis using as probe a cDNA expanding the C-terminal region (331 amino acids) of wheat Ppc1 gene. A single band was detected, which probably corresponded to transcripts from the different members of the PEPC gene family of wheat. To address the pattern of expression of Ppc1 gene, specific primers were designed, described in Section 2, to amplify a 171-bp fragment of the 3%-untranslated sequence of Ppc1 mRNA in Relative RT-PCR experiments (Fig. 5). This sensitive technique showed the presence of Ppc1 transcripts in any wheat organ or developmental stage analysed: developing seeds (Fig. 5A), aleurone and scutellum from germinating seeds (Fig. 5B), and roots and shoots from developing seedlings (Fig. 5C). Thus showing that Ppc1 gene is a ubiquitously expressed member of the wheat PEPC gene family. In spite of the wide expression of Ppc1 gene, it should be noted that during seed development a high accumulation of Ppc1 transcripts was detected at early stages (5 days) that progressively decreased to be almost undetectable at later stages of seed development (Fig. 5A). The western-blot analysis of PEPC polypeptides during wheat seed development showed the presence of an invariable 103- and a 108-kDa subunit, which was abundant at early stages of development and progressively disappeared at later stages [6], a pattern which matches the expression of Ppc1 gene (Fig. 5A). Since the PEPC polypeptide deduced from Ppc1 gene shows an expected molecular mass of 110 kDa, which fits reasonably well with the 108 kDa estimated for this PEPC subunit based on SDS-PAGE, it is likely that Ppc1 gene encodes the higher molecular weight PEPC polypeptide of wheat.
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Acknowledgements Thanks are due to Dr C. Hartman for generous gift of the wheat genomic library and to Dr Michael Hodges for in silico modelling of PEPC and helpful discussion of the manuscript. This work was supported by grant PB97-0745 from Direccio´ n General de Ensen˜ anza Superior, Ministerio de Educacio´ n y Cultura and Grant CVI 182 from Junta de Andalucı´a (Spain).