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First published online November 20, 2003; 10.1104/pp.103.030775 Plant Physiology 133:2021-2028 (2003) © 2003 American Society of Plant Biologists Structure and Expression of Phosphoenolpyruvate Carboxylase Kinase Genes in Solanaceae. A Novel Gene Exhibits Alternative Splicing1Plant Molecular Science Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom
Phosphorylation of phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) plays an important role in the control of central metabolism in higher plants. Two PPCK (PEPc kinase) genes have been identified in tomato (Lycopersicon esculentum cv Alicante), hereafter termed LePPCK1 and LePPCK2. The function of the gene products has been confirmed by transcription of full-length cDNAs, translation, and in vitro assay of kinase activity. Previously studied PPCK genes contain a single intron. LePPCK2 also contains a novel second intron that exhibits alternative splicing. The correctly spliced transcript encodes a functional PEPc kinase, whereas unspliced or incorrectly spliced transcripts encode a truncated, inactive protein. The relative abundance of the transcripts depends on tissue and conditions. Expression of LePPCK2 was markedly increased during fruit ripening. In ripe Alicante fruit, the locule and seeds contained only the correctly spliced LePPCK2 transcripts, whereas in ripe fruit of the tomato greenflesh mutant, they contained correctly and incorrectly spliced transcripts. Potato (Solanum tuberosum) contains genes that are very similar to LePPCK1, and LePPCK2; StPPCK2 exhibits alternative splicing. Aubergine (Solanum melongena) and tobacco (Nicotiana tabacum) also contain a PPCK2 gene; the sequence of the alternatively spliced intron is highly conserved between all four species. The data suggest that the two PPCK genes have different roles in tissue-specific regulation of PEPc and that the alternative splicing of PPCK2 transcripts is functionally significant.
Phosphoenolpyruvate carboxylase (PEPc; EC 4.1.1.31) is a ubiquitous enzyme in higher plants. It catalyzes the carboxylation of phosphoenolpyruvate to form oxaloacetate and inorganic phosphate and plays a range of roles in different tissues. In C4 and Crassulacean acid metabolism (CAM) plants, a photosynthetic isoform of PEPc catalyzes the primary fixation of CO2 (O'Leary, 1982  ; Andreo et al., 1987). In most non-photosynthetic tissue and in C3 leaves, PEPc is the major anapleurotic enzyme. It allows the replenishment of tricarboxylic acid cycle intermediates to provide the precursors for biosynthetic pathways such as amino acid biosynthesis. It also provides malate in guard cells, legume root nodules, and developing fruit (Andreo et al., 1987; Chollet et al., 1996; Vidal and Chollet, 1997). To fulfill these different functions, PEPc is encoded by a small gene family whose members are expressed in different types of tissue (Lepiniec et al., 1994).
PEPc is strongly regulated by pH and by positive and negative effectors (Glc 6-phosphate and malate, respectively; Andreo et al., 1987
After the cloning of PEPc kinase genes (termed PPCK), it has become clear that PEPc kinase comprises a Ser/Thr kinase catalytic domain with essentially no extensions. It resembles the protein kinase catalytic domain of plant calcium-dependent protein kinases but lacks the C-terminal Ca2+-binding EF hands and the N-terminal extensions of these enzymes (Hartwell et al., 1999a
Recent work in the model C3 species Arabidopsis showed the existence of two isoforms of PEPc kinase with different expression patterns (Fontaine et al., 2002 Here, we report on the cloning and expression of two isoforms of PEPc kinase from tomato. The data show one unique feature of PEPc kinase in the Solanaceae. All PEPc kinase genes reported to date contain just one intron, close to the 3' end of the coding sequence. However, one of the PEPc kinase genes of tomato contains a novel second intron, which is subject to alternative splicing. One transcript encodes a functional PEPc kinase, but two transcripts encode a truncated, inactive protein. Our results show that the relative abundance of the transcripts is dependent on tissue and conditions. Similar genes, also subject to alternative splicing, have been detected in potato (Solanum tuberosum), tobacco (Nicotiana tabacum), and aubergine (Solanum melongena).
Identification of LePPCK1 and LePPCK2 An examination of the expressed sequence tag (EST) database suggested that tomato contains two PPCK genes. One putative full-length PEPc kinase EST (GenBank accession no. AW033195), hereafter termed LePPCK1, was completely sequenced (Gen-Bank accession no. AF203481). The deduced amino acid sequence (279 residues, 31.4 kD) is aligned with several other PEPc kinase sequences in Figure 1. To examine the structure of the LePPCK1 gene, we designed PCR primers (see Table I) to amplify genomic DNA. A single band of 1148 bp was generated, cloned, and sequenced (GenBank accession no. AY190084). Inspection of this sequence showed that the coding sequence comprises two exons, with one 89-bp intron from base 808 to 896 of AY190084 inclusive (see Fig. 2).
The LePPCK1 cDNA was subcloned behind a T3 promoter in pBluescript. To test whether the full-length cDNA was functional, the plasmid was linearized with NotI, transcribed, and translated. The translation product was then assayed for PEPc kinase activity. Figure 3 shows that the translation product was of the expected size (31 kD) and was able to phosphorylate PEPc.
A second putative full-length tomato PEPc kinase EST (GenBank accession no. AW223421), hereafter termed LePPCK2, was also completely sequenced (data not shown; see below). Inspection of this sequence showed that the cDNA contains an insertion of 138 bp near the middle of the coding region relative to other PEPc kinase cDNAs. The insertion also included an in-frame stop codon. To examine the structure of the gene, PCR primers were designed to amplify cDNA (from tomato fruit) and genomic DNA, and the products were sequenced. Analysis of these sequences revealed that the LePPCK2 gene (GenBank accession no. AY188444) contains two introns (Fig. 2). Like other PEPc kinase genes, one is located close to the 3' end of the coding sequence (bases 1,044-1,212 of AY188444). The second intron (bases 372-604 of AY188444) is located close to the middle of the coding region and possesses two alternative 5' splice start sites (bases 372 and 510, see Fig. 2). The in-frame stop codon is between these sites (bases 450-452 of AY188444). Hence, splicing can give rise to a transcript that encodes a functional PEPc kinase (transcript 1, the full sequence of which is GenBank accession no. AY187634), an incorrectly spliced transcript with a premature stop codon (transcript 2), and an unspliced transcript with a premature stop codon (transcript 3). Transcripts 2 and 3 both encode a truncated, nonfunctional PEPc kinase. As shown in Table II, ESTs corresponding to each transcript have been detected. The deduced amino acid sequence encoded by transcript 1 (278 residues, 31.1 kD) is shown in Figure 1. There is 61% sequence identity between the two tomato PPCK proteins.
LePPCK2 transcript 1 was positioned behind a T7 promoter in pCR4-TOPO, and LePPCK2 transcript 2 was positioned behind a T3 promoter in pBluescript. The clones were linearized, transcribed, and translated. The translation products were then assayed for PEPc kinase activity. Figure 3 (lane 3) shows that the product from transcript 1 was of the expected size (31 kD) and was able to phosphorylate PEPc as effectively as did K. fedtschenkoi PEPc kinase generated in a similar way (lane 5). Transcript 2 consistently directed the synthesis of a 16-kD protein (compare Fig. 3, lane 4 with the no RNA control lane 6 in A) that did not phosphorylate PEPc. The size of this protein was as expected from the position of the in-frame stop codon in LePPCK2 transcript 2. A slightly smaller band can be seen in the translation products from the full-length PEPc kinase clones (lanes 1, 3 and 5). This presumably results from either breakdown of the major product or premature termination of translation.
PEPc kinase is expressed at a low level in C3 plants, with the exception of legume root nodules (Nakagawa et al., 2003
In view of the developmental control of expression exhibited by LePPCK2 and the positional control of splicing shown in Figures 4 and 5A, we examined the expression of this gene in the gf (greenflesh) tomato mutant, in which the breakdown of chlorophyll during fruit ripening is markedly reduced (Akhtar et al., 1999
Further analysis of the EST database revealed two putative potato PEPc kinase genes. As summarized in Table II, several ESTs closely resemble LePPCK1; none of these, however, are full length. Other ESTs resemble either transcript 2 or 3 of LePPCK2. To examine the structure of the corresponding genes, hereafter termed StPPCK1 and StPPCK2, we designed PCR primers (see Table I) to amplify cDNA (from mature leaves) and genomic DNA. Using StPPCK1 primers, a partial genomic sequence of 913 bp was cloned and sequenced (GenBank accession no. AY219178); this contains only one intron as for LePPCK1 (bases 765-877 of AY219178). Comparison of the deduced amino acid sequences of StPPCK1 and LePPCK1 shows 98.5% identity over 265 amino acids (see Fig. 1). To test whether StPPCK2 exhibits alternative splicing, we designed primers to the region flanking its putative additional intron based on the sequence of the ESTs BG594065 and BG594668. Using these, we amplified three bands of the predicted mobilities from potato leaf cDNA but only one from genomic DNA (Fig. 6A), indicating that StPPCK2 does contain an additional intron that is subject to alternative splicing. The sequence of the partial genomic fragment of StPPCK2 (555 bp, GenBank accession no. AY293738) matches the sequences of the ESTs corresponding to StPPCK2 (Table II) almost exactly (not illustrated).
To check other members of the Solanaceae, we amplified and sequenced a 553-bp fragment of aubergine genomic DNA (GenBank accession no. AY236482) and a 538-bp fragment of tobacco genomic DNA (GenBank accession no. AY347261) using the primers designed for RT-PCR of LePPCK2 (Table I) that span the unusual intron in LePPCK2. The resulting sequences are very similar to the sequences of the LePPCK2 and StPPCK2 genes. Figure 7 shows a comparison of the intron sequences, which comprise 233, 234, 232, and 217 bp in tomato, potato, aubergine, and tobacco, respectively. The two donor splice sites and the acceptor site are conserved. All four sequences contain at least one in-frame stop codon 70 to 80 bp into the intron (bold in Fig. 7); aubergine has two closely adjacent stop codons. The tobacco intron has a deletion of 18 bp relative to the other introns just 3' to the second ("incorrect") donor splice site. Using the same primers, we amplified three bands of the predicted mobilities from tobacco flower and leaf cDNA but only one from genomic DNA (Fig. 6B), indicating that the tobacco PPCK2 gene does contain an additional intron that is subject to alternative splicing. As with tomato, the relative abundance of the three transcripts differs between organs (Fig. 6B). The data argue strongly that both aubergine and tobacco contain a gene equivalent to the PPCK2 genes of potato and tomato.
The results presented here demonstrate that tomato contains at least two genes that encode functional PEPc kinases. LePPCK1 is similar to other reported PEPc kinase genes (Hartwell et al., 1999a ; Taybi et al., 2000; Tsuchida et al., 2001; Fontaine et al., 2002; Nakagawa et al., 2003; Nimmo, 2003; Xu et al., 2003) in that it encodes a protein kinase catalytic domain with minimal N- and C-terminal extensions and contains one small intron toward the 3' end of the coding region. LePPCK2 has one major difference from other PPCK genes studied to date, namely the presence of an additional intron that exhibits alternative splicing. Our data show that three types of transcript can be detected, corresponding to correctly spliced, incorrectly spliced, and unspliced transcripts. The correctly spliced transcript encodes a functional PEPc kinase very similar to the enzyme from other species, whereas the other two encode truncated, nonfunctional proteins. It remains to be seen whether the truncated product accumulates in cells. A similar gene has been detected in potato, aubergine, and tobacco (Figs. 6 and 7). To date, this unusual PPCK gene with alternative splicing has been found only in the Solanaceae.
Several lines of evidence suggest that this alternative splicing of PPCK2 transcripts may be functionally significant. First, the nucleotide sequence of the intron is highly conserved between four members of the Solanaceae (see Fig. 7); moreover, the intron and exon sequences are equally similar. For example, the aubergine and tomato sequences are 94% identical over 321 bases of exon sequence (not shown) and 92% identical over 233 intron bases (Fig. 7). The conservation of intron sequence in the PPCK2 genes of the four species includes the splice sites as shown both directly (Fig. 7) and functionally (Fig. 6); it also covers the presence of in-frame stop codons that would lead to premature truncation of the gene product (although in aubergine there are actually two in-frame stop codons as shown in Fig. 7). The major difference between the intron sequences is an 18-bp deletion in the tobacco sequence that is downstream of both the internal stop codon and the "incorrect" donor splice site. Second, as noted below, the relative abundance of the three transcripts in tomato depends on tissue and conditions. Third, transcripts 2 and 3 actually predominate in some samples even though they contain a premature in-frame stop codon, which often leads to instability of the mRNA (Abler and Green, 1996
Various possible roles for this alternative splicing can be envisaged. First, it could allow control of the abundance of the functional transcript 1, either in the context of tissue specificity or in response to a signal; a complex example of such behavior is found in the alternative processing of the Arabidopsis FCA gene (Macknight et al., 2002
It is possible that cells contain limiting amounts of a splicing factor that is essential for the production of transcript 1; if so, roots must contain more of this factor than, for example, leaves. Requirement for a specific factor to give correct splicing would be consistent with the sequence of the intron. Plant introns are AU and particularly U rich (Brown and Simpson, 1998
In a second potential role of the alternative splicing, it is possible that the truncated protein expressed in vitro from LePPCK2 transcripts 2 and 3 (Fig. 3) may accumulate in vivo and play a functional role. This truncated protein would extend just beyond the N-terminal ATP-binding domain of PEPc kinase; it may be able to fold stably, to bind ATP, and/or to interact with other proteins such as PEPc. Hence, this truncated protein could play an unsuspected role in the control of the phosphorylation state of PEPc. It clearly will be important to assess whether the truncated protein does accumulate in cells. However, it must be pointed out that PEPc kinase is a very low-abundance protein. For example, even in highly illuminated maize leaves, a rich source, it comprises less than 1 in 106 of soluble protein (Saze et al., 2001
To gain information about the possible functions of the two tomato PPCK genes, we examined their tissue expression patterns. Our RT-PCR data agree with the distribution of ESTs between tissues (Table II); both approaches show that LePPCK1 is expressed in many organs, whereas LePPCK2 is predominantly expressed in ripening fruit. This would suggest that LePPCK1 encodes a housekeeping kinase, mainly involved in ensuring the replenishment of the TCA cycle, whereas the main role of the kinase encoded by LePPCK2 is in late ripening. ESTs corresponding to the two potato PPCK genes are found in different libraries (Table II). Following the arguments of Ronning et al. (2003
Recently, Guillet et al. (2002
Plant Material Tomato (Lycopersicon esculentum cv Alicante) plants were grown from seed in bedding compost (William Sinclair Horticulture Ltd., Lincoln, UK) in a greenhouse under a 16-h photoperiod supplemented with mercury vapor lamps (300 µmol m-2 s-1). Plants were repotted at 4 weeks and were watered every 2 d. Tomato fruits were allowed to ripen on the vine. RNA was isolated from quartered segments. For localization studies, fruit were collected at the ripe red stage and separated into skin, outer pericarp, inner pericarp, locule, and seeds. Young leaves were harvested as the primary leaf 14 d after sowing, whereas mature leaves were harvested after 6 weeks. Seedlings (8 d old) were harvested after either 10 h of darkness or 3 h of light.
Plant material was frozen under liquid nitrogen and stored at -70°C. Frozen plant tissue (2-3 g) was ground to a fine powder using an autoclaved mortar and pestle. RNA was then isolated according to the protocol of Chang et al. (1993
Genomic DNA was extracted using a DNA isolation kit (PUREgene DNA isolation kit, Gentra Systems, Gentra Systems, Minneapolis). The quantity and purity of the DNA was determined spectrophotometrically according to the method described by Sambrook et al. (1989
The RNA samples (100 ng) were mixed with 0.25 µM oligo(dT) for 5 min at 70°C and cooled at 4°C for 5 min. RT was carried out in a reaction mixture (50 µL) containing avian myeloblastosis virus reverse transcriptase buffer, 1 mM dNTPs, 1 unit µL-1 RNase inhibitor, and 0.4 units µl-1 avian myeloblastosis virus reverse transcriptase (all from Promega, Madison, WI). The reaction was performed at 48°C for 45 min. The enzyme was then heat inactivated at 95°C for 5 min, and the samples were used directly for PCR. PCR reactions were performed using 2.5 µL of each cDNA sample in a reaction mixture (25 µL) containing 12.5 µL of 2x Reddy Mix (Abgene, Epsom, UK) and 0.5 µM of the 5' and 3' primers. The primer sequences are shown in Table I. The PCR reactions were conducted in a programmable thermocycler (PCR Sprint, Hybaid, Ashford, UK). The reaction conditions for the amplification of LePPCK1 were an initial denaturation step of 94°C for 5 min, 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 60 s, and a final extension step of 72°C for 5 min. The reaction conditions for the amplification of LePPCK2 were an initial denaturation step of 94°C for 5 min, 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and a final extension step of 72°C for 5 min. Primers for either Actin52 or ubiquitin were used as a constitutive control in both conditions. After amplification, reactions were resolved by electrophoresis on a 1% (w/v) agarose gel and stained with ethidium bromide.
PCR fragments were extracted from an agarose gel by purifying with QIAquik gel extraction kit (Qiagen USA, Valencia, CA) and cloned directly into pCR4-TOPO vectors using the TOPO TA cloning kit with one shot cells (Invitrogen, Carlsbad, CA). All clones were sequenced using universal primers (MWG-Biotech, Ebersberg, Germany).
This was carried out as described previously (Fontaine et al., 2002
We thank Prof. Don Grierson for the gift of gf tomato seeds. Received July 25, 2003; returned for revision August 19, 2003; accepted September 5, 2003.
1 This work was supported by the Biotechnology and Biological Sciences Research Council (PhD studentships to J.T.M. and S.S. and research support to H.G.N.). 
2 Present address: Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, UK. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030775. * Corresponding author; e-mail h.g.nimmo{at}bio.gla.ac.uk; fax 44-141-330-4620.
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