Plant Physiol. (1999) 120: 1033-1042

Phosphoenolpyruvate Carboxykinase in the C₄ Monocot Urochloa panicoides Is Encoded by Four Differentially Expressed Genes¹

Patrick M. Finnegan^*, Shoichi Suzuki, Martha Ludwig, and James N. Burnell

Division of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australian Capital Territory 0200, Australia (P.M.F.); Japan Tobacco, 700 Higashibara Toyota-Cho Iwata Gun, Shizuoka 438-0802, Japan (S.S.); Department of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia (M.L.); and Department of Biochemistry and Molecular Biology, James Cook University, Townsville, Queensland 4810, Australia (J.N.B.)

	ABSTRACT

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Previous screening of a cDNA library of leaf poly(A⁺) RNA from Urochloa panicoides, a phosphoenolpyruvate carboxykinase (PCK)-type C₄ monocot, led to the characterization of cDNAs encoding the U. panicoides PCK subunit PCK1. A second PCK sequence, designated PCK2, has now been found by rescreening the library. The deduced PCK2 polypeptide is 626 residues in length, has a predicted molecular mass of 68,686 D, and is 96% identical to the deduced PCK1 sequence. Isolation and characterization of genomic DNA fragments revealed that the PCK1 and PCK2 genes are each closely linked to another PCK gene. These additional genes have been designated PCK3 and PCK4, respectively. In each case, the second gene is located upstream and in the same transcriptional orientation as the gene characterized through cDNA analysis. A reverse transcription-polymerase chain reaction assay was used to demonstrate that PCK1 and PCK2 transcripts predominate in leaves, whereas PCK3 and PCK4 transcripts predominate in roots. Moreover, accumulation of PCK1 and PCK2 transcripts is light dependent. Direct N-terminal sequencing of PCK polypeptides purified from leaves demonstrated that PCK2 is produced. These results strongly suggest that PCK1 and PCK2 are involved in the photosynthetic CO₂-concentrating mechanism active in U. panicoides.

	INTRODUCTION

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PCK (ATP [GTP]: oxaloacetate carboxylase [transphosphorylase], EC 4.1.1.32 [GTP dependent] or EC 4.1.1.49 [ATP dependent]) is widespread in nature and catalyzes the reversible decarboxylation of oxaloacetate to PEP. The GTP-dependent enzyme found in animals catalyzes the first committed step in gluconeogenesis (Utter and Kolenbrander, 1972). The ATP-dependent enzyme of plants performs this function in the cotyledons of species with fat-storing seeds, mobilizing reduced carbon from lipids for use in other tissues of the seedling (Leegood and ap Rees, 1979). The plant enzyme also has a key role in photosynthetic carbon assimilation in one group of C₄ (Hatch, 1987) and CAM (Dittrich et al., 1973) plants, and in some species of algae (Reiskind and Bowes, 1991), and may be involved in the response of Brassica napus to chilling (Sáez-Vásquez et al., 1995).

In PCK-type C₄ grasses such as Urochloa panicoides, PCK is involved in the carbon-concentrating mechanism inherent in photosynthetic tissues (Hatch, 1987). Located in the cytosol (Ku et al., 1980; Chapman and Hatch, 1983), PCK is the major decarboxylating enzyme found in the bundle sheath cells (Hatch, 1987). Through the decarboxylation of oxaloacetate to PEP, PCK helps raise the CO₂ concentration in the bundle sheath cells to levels much higher than that found in mesophyll cells or the atmosphere. The increased level of CO₂ then suppresses photorespiration in these plants (Hatch, 1987).

The plant PCK is a multimeric enzyme of identical subunits (Burnell, 1986; Walker et al., 1995). In PCK-type C₄ plants, the leaf enzyme involved in photosynthesis is hexameric (Burnell, 1986). In contrast, the enzyme found in gluconeogenic cucumber (Cucumis sativus) cotyledons is tetrameric (Walker and Leegood, 1995; Walker et al., 1995). The full-length U. panicoides PCK subunit is 68 kD (Finnegan and Burnell, 1995), but the subunit size varies from 67 to 78 kD in other species (Walker et al., 1995; Walker and Leegood, 1996). The N terminus of the plant PCK subunit may contain regions important for enzyme regulation, because it contains a target site for dark-dependent, light-reversible phosphorylation in most species examined (Walker and Leegood, 1996; Walker et al., 1997). The enzyme from the leaves of U. panicoides and several other C₄ grasses is not susceptible to phosphorylation (Walker and Leegood, 1996). However, the N terminus of the leaf subunit is extremely labile (Finnegan and Burnell, 1995; Walker et al., 1995), which is true for the enzyme subunit from all species examined (Walker and Leegood, 1996; Walker et al., 1997).

A single complete cDNA sequence for a PCK subunit has been reported for cucumber (Cucumis sativus) (Kim and Smith, 1994) and the PCK-type C₄ grasses U. panicoides (Finnegan and Burnell, 1995), Spartina anglica (accession no. E12730), and Zoysia japonica (accession no. E12731). However, genome sequencing of Arabidopsis has revealed three possible PCK gene sequences (accession nos. AC004705, CAA16690, and CAB38935). Moreover, analysis of U. panicoides cDNAs (Finnegan and Burnell, 1995) indicated that the enzyme subunit is also encoded by a multigene family in this species. Northern analysis using the U. panicoides PCK1 cDNA as a probe showed that the accumulation of PCK transcripts in dark-grown U. panicoides seedlings is induced by light (Finnegan and Burnell, 1995), which is in keeping with the role of the enzyme in photosynthesis. We report the cDNA sequence corresponding to a second U. panicoides PCK gene, PCK2, as well as the partial genomic characterization of two other PCK genes, PCK3 and PCK4. Results of a RT-PCR assay indicated that PCK1 and PCK2 are expressed in a leaf-predominant manner, and are therefore likely to be the subunits involved in photosynthesis. In contrast, transcripts from PCK3 and PCK4 accumulate predominantly in roots.

	MATERIALS AND METHODS

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Plant Material

2

1

Library Construction and Screening

View larger version (11K): [in this window] [in a new window]   Figure 1. Cloning of cDNAs encoding U. panicoides PCK2. A cDNA library derived from U. panicoides leaf poly(A+) RNA was screened by hybridization with progressively more 5 fragments of U. panicoides PCK cDNA until a full-length PCK2 sequence was obtained. A schematic diagram of the composite PCK2 cDNA is shown above the constituent cDNA fragments. The ORF (box) and the direction of transcription (arrow) for PCK2 are indicated. The length and position of the PCK1 cDNA used as the hybridization probe in Southern analysis is also shown.

Southern-Blot Analysis

1

Oligonucleotide Primers

RT-PCR Assays

Miscellaneous Methods

	RESULTS

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Multiple Genes Encode PCK in U. panicoides

When compared over their entire lengths, the PCK1 and PCK2 cDNAs were 94.4% identical at the nucleotide level. Much of the variation occurred in the 3 UTRs of the cDNAs, which were 82.5% identical (Fig. 2). The variation between the 3 UTRs was mainly due to a 26-bp insertion in PCK1 that includes one copy of a 23-bp direct repeat. The PCK2 cDNA has an additional 44-bp 3 extension compared with PCK1.

View larger version (29K): [in this window] [in a new window]   Figure 2. Comparison of the nucleotide sequences at the 3 ends of four putative U. panicoides PCK mRNAs. The sequence of the PCK1 cDNA (accession no. U09241) from the SacI site (bold) located 234 bp upstream of the stop codon (double underline) to the poly(A+) addition site is shown on the top line. The homologous sequences of the PCK2 cDNA (accession no. AF136161) and the PCK3 (accession no. AF136162) and PCK4 (accession no. AF136163) genes are shown below. Dots represent residues identical to PCK1 and dashes denote gaps introduced to optimize the alignment. The position of the single intron covered by this comparison and conserved in all four U. panicoides PCK genes is indicated by the vertical arrowhead. The intron sequences have been omitted from this comparison. The position and direction of the "universal" PCK primers UP1 and UP2 (horizontal arrows) and the sequences to which primers specific for each PCK gene anneal (underlines) are shown. The 23-bp repeat sequences in the PCK1 3 UTR are shown in bold italics.

The deduced PCK2 polypeptide had 626 amino acid residues (Fig. 3), two more than the PCK1 polypeptide, and a calculated molecular mass of 68,686 D. The latter corresponds exactly to the estimated size of the U. panicoides protein obtained from immunoblots (68-69 kD; Finnegan and Burnell, 1995; Walker et al., 1995). The PCK2 ORF had 74 nucleotide differences compared with that of PCK1. This variation gave rise to 26 amino acid differences (Fig. 3), of which 21 (81%) were nonconservative. The differences between the U. panicoides PCK1 and PCK2 polypeptides were mainly localized to the N- and C-terminal regions of the proteins. Only one difference, the conservative substitution of Thr-236 in PCK1 for a Ser in PCK2, occurred within any of the subsequences previously identified as having above-average similarity among ATP-dependent PCKs (Linss et al., 1993; Finnegan and Burnell, 1995). Moreover, none of the differences involved residues thought to be present in the PCK active site as defined by analysis of the crystal structure of the E. coli enzyme (Fig. 3; Matte et al., 1996).

View larger version (48K): [in this window] [in a new window]   Figure 3. Comparison of plant PCK subunit sequences. The entire U. panicoides PCK1 amino acid sequence is shown on the top line in the single-letter code. Residues present in the PCK active site (Matte et al., 1996) are underlined. N-terminal residues determined directly from PCK polypeptides are overlined and are assigned Roman numerals corresponding to the polypeptides in Figure 8. Dots in the U. panicoides (Up) PCK2, S. anglica (Sa), Z. japonica (Zj), cucumber (Cs), and Arabidopsis (At) subunit sequences indicate residues found in the U. panicoides PCK1 sequence. Gaps (dashes) were introduced to maximize identity. The numbering refers to the positions within the comparison.

The complete primary structure of a single PCK subunit has been determined for cucumber (Kim and Smith, 1994) and the PCK-type C₄ monocots S. anglica (accession no. E12730) and Z. japonica (accession no. E12731), whereas genes for two complete subunits have been characterized for Arabidopsis (accession nos. CAA16690 and CAB38935). All of these proteins had N-terminal extensions compared with the PCKs of U. panicoides, ranging from 29 residues for the Z. japonica subunit to up to 48 residues for one of the Arabidopsis subunits (Fig. 3). Within the overlapping sequences (Fig. 3), the S. anglica and Z. japonica PCKs were 80% identical to U. panicoides PCK1, while the cucumber and Arabidopsis subunits were 75% identical to this subunit. Many of the differences among the subunits were located in the N-terminal portion of the comparison (Fig. 3). When the comparison was restricted to the sequences following Ser-95 of U. panicoides PCK1, the identity with U. panicoides PCK1 increased to 90% for the S. anglica and Z. japonica subunits and to nearly 85% for the cucumber and Arabidopsis sequences.

Genomic Organization of PCK Genes

View larger version (8K): [in this window] [in a new window]   Figure 4. Organization of PCK genes in U. panicoides. Schematic representations of the SalI restriction fragment maps of two genomic bacteriophage lambda clones encoding the four identified PCK genes in U. panicoides are shown and include the ORF (boxes) and the direction of transcription (arrows) for each gene. The SalI fragments that hybridize to a 1.4-kb partial PCK1 cDNA (see Fig. 1) are indicated with asterisks. The dashed line extends to a SalI site mapped by genomic Southern analysis.

The possibility that U. panicoides may have more than four PCK subunit genes was examined by genomic Southern hybridization analysis (Fig. 5). Restriction digests of U. panicoides total cellular DNA were separated on an agarose gel, transferred to a membrane, and probed with the 1.4-kb PCK100101 cDNA insert covering the C-terminal 60% of the ORF and the entire 3 UTR of the PCK1 mRNA (Fig. 1). This probe detected five fragments in SalI digests of U. panicoides genomic DNA (Fig. 5, lane 1). Examination of the maps of clones 8-1 or 13-1 (Fig. 4) indicated that each of these labeled fragments corresponded to a SalI fragment predicted to hybridize to the probe. The 10-kb hybridizing fragment corresponded in size to the fragment extending from the C-terminal end of PCK3 into the N-terminal portion of PCK1 on the map of 13-1, whereas the 3.3-kb hybridizing fragment spanned the C terminus of PCK1. The 1.2- and 14-kb hybridizing fragments were located on 8-1; the 1.2-kb fragment was entirely within PCK4. The 14-kb fragment extended from within the C-terminal portion of PCK4, across the PCK4-PCK2 intergenic region, and into the N-terminal part of PCK2. From the maps of the 8-1 and 13-1 inserts, the only other fragment larger than 500 bp expected to hybridize to the probe would encode the C terminus of PCK2 and would be greater than 1.2 kb. It is likely, then, that the 8-kb hybridizing fragment corresponded to this fragment. Due to the high-stringency wash conditions used, the variation in hybridization signals among the bands probably reflects sequence divergence among the PCK genes. In this regard, the 3.3-kb SalI fragment, which contains sequences identical to the probe, produced the strongest signal.

View larger version (76K): [in this window] [in a new window]   Figure 5. Genomic Southern analysis of PCK genes. Approximately 10 µg of U. panicoides total genomic DNA was digested with the indicated restriction endonucleases. The fragments were separated on a 0.7% (w/v) agarose gel, transferred to a nylon membrane, and probed with a 1.4-kb U. panicoides PCK1 partial cDNA (Fig. 1). The positions and sizes (in kb) of the HindIII fragments of bacteriophage lambda are shown on the left as markers.

When the genomic DNA digested with PstI, EcoRI, BglII or BamHI (Fig. 5, lanes 2-5) was probed with the 1.4-kb PCK1 cDNA fragment, relatively simple hybridization patterns were obtained (Fig. 5). In both the BglII and BamHI digests, only three hybridizing fragments were observed. Given the length of the PCK3-PCK1 (7.5 kb) and PCK4-PCK2 (11 kb) intergenic regions, and the fact that the coding regions for PCK1 and PCK2 were each about 3.5 kb (Fig. 4), it is unlikely that the hybridizing 20-kb BamHI fragment would have segments of more than two PCK genes. The 4.9- and 3.4-kb BamHI fragments were each too small to carry more than one PCK gene. Similarly, the 17-kb BglII fragment is unlikely to span more than two PCK genes, whereas the 8.5- and 7.9-kb fragments were not large enough to span the distance between regions known to hybridize to the probe. Taken together, these observations indicate that the PCK multigene family contains only four closely related members, but the possibility of other more divergent members cannot be eliminated.

3-End Sequence Analysis of the PCK Genes

Tissue-Dependent Expression of PCK Genes

View larger version (63K): [in this window] [in a new window]   Figure 6. Tissue-specific expression of U. panicoides PCK genes. A, RT-PCR assay using universal PCK primers. The universal PCK primer UP1 (see Fig. 2) was used to prime reverse transcription of 1 µg of total RNA isolated from leaves (lanes 1 and 2) or roots (lanes 3 and 4) of a light-grown plant. The reactions either contained (lanes 1 and 3) or lacked (lanes 2 and 4) reverse transcriptase. The products were amplified by PCR using the universal PCK primers UP1 and UP2 (see Fig. 2) before separation on an agarose gel. B, Semi-nested RT-PCR using PCK-specific primers (see Fig. 2). The RT-PCR products from A were purified, diluted 105-fold, and reamplified by standard PCR (see ``Materials and Methods''). The products obtained from the reamplification of the root (lanes 1-5) or leaf (lanes 6-10) RT-PCR products are shown. The semi-nested PCR step was primed with universal primer UP2 and the primers specific for PCK1 (lanes 1 and 6), PCK2 (lanes 2 and 7), PCK3 (lanes 3 and 8), or PCK4 (lanes 4 and 9). Competitive PCRs containing primer UP2 and all four gene-specific primers are also shown (lanes 5 and 10). The positions and sizes (in kb) of the AvaII fragments of bacteriophage lambda are shown on the left as markers.

When total RNA from the green leaves of U. panicoides was subjected to RT-PCR, two predominant products of 435 and 405 bp were detected (Fig. 6A, lane 1) corresponding in size to the products expected from PCK1 and PCK2 transcripts, respectively. Two additional products of about 490 and 380 bp were also obtained. The origins of these products are not known, but neither hybridized to PCK1 cDNA probes (not shown). Total RNA isolated from root tissue gave rise to a single product band of about 400 bp (Fig. 6A, lane 3) that was indistinguishable in size from the 393- and 396-bp products expected from PCK3 and PCK4 transcripts, respectively. None of the RT-PCR products observed in this experiment were due to genomic DNA contamination of the leaf and root RNA preparations, because no products were detected when reverse transcriptase was omitted from the assay (Fig. 6A, lanes 2 and 4). The results of this experiment indicate that PCK1 and PCK2 are expressed in a leaf-predominant manner, whereas some combination of PCK3 and PCK4 is expressed in a root-predominant manner.

As the RT-PCR products from PCK3 and PCK4 transcripts could not be distinguished from one another by length or restriction site polymorphisms due to the high similarity of the 3 regions of these genes (Fig. 2), a semi-nested PCR assay was employed to examine their expression. The UP1/UP2 RT-PCR products from leaf and root RNA were purified and re-amplified using the universal primer UP2 together with individual primers specific for each PCK gene (Fig. 2). Each primer pair yielded only the single predicted product when used to amplify the RT-PCR product from leaf and root RNA (Fig. 6B). These products were 314 bp for PCK1 (lanes 1 and 6), 308 bp for PCK2 (lanes 2 and 7), 231 bp for PCK3 (lanes 3 and 8), and 273 bp for PCK4 (lane 4). The only variation in this pattern was that the UP2/PCK4-specific primer pair did not always produce a product when leaf RNA was the starting material (Fig. 6B, lane 9).

The relative abundance of the products from these four reactions was dependant on the tissue source of the RNA template. When leaf RNA was tested, similar amounts of the PCK1 and PCK2 products were obtained. These amounts were consistently greater than those of the PCK3 and PCK4 products produced (Fig. 6B, compare lanes 6 and 7 with lanes 8 and 9). This result is similar to that seen for the direct RT-PCR experiment described above, and suggests that transcripts from the former two genes are more abundant in leaf tissue than transcripts from the latter two genes. However, when root RNA was examined, PCK1- and PCK2-specific products were always obtained in much lower amounts than the PCK3 and PCK4 products (Fig. 6B, compare lanes 1 and 2 with 3 and 4), implying that the transcripts from the latter two genes are more abundant in root tissue than PCK1 and PCK2 transcripts.

The qualitative differences observed when the RT-PCR products were amplified with the gene-specific primers in separate reactions was verified by semicompetitive PCR. Root RNA was amplified in a semi-nested RT-PCR assay in which primer UP2 and all four gene-specific primers were combined in a single reaction. This produced only the PCK3- and PCK4-specific products in similar amounts (Fig. 6B, lane 5). These products were not detected when leaf RNA was the template (Fig. 6B, lane 10); instead, two different products, neither of which was evident when root RNA was tested, were obtained. The 310-bp species is a doublet of the PCK1- and PCK2-specific products. The other product, with slightly lower mobility, was of unknown origin and only arose when the four gene-specific primers were combined. Therefore, this product probably arose from amplification of an unknown template through priming by two of these primers. Although the PCR assays described here are not quantitative, the results strongly indicate that the PCK1 and PCK2 transcripts are the most abundant in leaf tissue and that PCK3 and PCK4 transcripts are the most abundant in root tissue.

Light Induction of Individual PCK Genes

View larger version (39K): [in this window] [in a new window]   Figure 7. Light induction of U. panicoides PCK genes. Total RNA (1 µg) isolated from the roots (lane 1) or leaves (lane 8) of a light-grown plant or from the cotyledons of 7-d-old dark-grown plants illuminated with 100 µmol quanta m2 s1 light for 0 (lane 2), 6 (lane 3), 12 (lane 4), 24 (lane 5), 30 (lane 6), or 36 (lane 7) h just prior to harvesting was subjected to RT-PCR using the universal PCK primers UP1 and UP2, as described in the legend to Figure 6A. The positions and sizes (in kb) of the AvaII fragments of bacteriophage lambda are shown on the left as markers.

When RNA from dark-grown shoots was tested for the presence of PCK transcripts, products were only obtained for one of six individuals. These products were identical to those from RNA from green leaves (Fig. 7, lane 2), but the band intensities were lower than normally observed using 1 µg of leaf total RNA (Fig. 7, lane 8). After 6 h of illumination, the PCK RT-PCR pattern in all six dark-grown cotyledons tested was exactly the same as in a green leaf (Fig. 7, compare lane 3 with lane 8). This pattern did not change with continuous illumination for 12 to 36 h (Fig. 7, lanes 4-7). The lack of detectable RT-PCR products from five of six dark-grown shoots was probably not due to failure of the assay, because RT-PCR products were obtained from all 23 light-exposed cotyledons examined; rather, it suggests a low abundance of PCK transcripts in dark-grown shoots.

Presence of PCK1 and PCK2 in Leaves

View larger version (30K): [in this window] [in a new window]   Figure 8. N-terminal amino acid analysis of U. panicoides PCK polypeptides. PCK was purified from U. panicoides leaves using a method that results in the stepwise cleavage of up to 8 kD from the N terminus of a high proportion of the polypeptide chains (Finnegan and Burnell, 1995). The mixture of cleavage products was subjected to Edman degradation. The amino acid residues released in each cycle are shown in the matrices and are arranged in order of decreasing abundance (at the top of each column is the most abundant residue released). The sequences below the line in each matrix were deduced from the PCK1 and PCK2 cDNAs. The Roman numerals correspond to the sequences previously identified (Finnegan and Burnell, 1995). In the body of each matrix, residues found in both PCK1 and PCK2 sequences are indicated by white boxes, whereas residues found in only the PCK1 or the PCK2 sequence are indicated by shaded and white circles, respectively. This analysis was also performed with the SDS-PAGE-purified 62- and 61-kD PCK degradation products (Finnegan and Burnell, 1995).

In the previous study (Finnegan and Burnell, 1995), the N-terminal sequences present in the isolated 62- and 61-kD PCK fragments were also determined. Re-analysis of these results supported the conclusion that both PCK1 and PCK2 are produced within leaves (Fig. 8). The 62-kD band clearly contained N-terminal sequences III and IV derived from PCK2 and PCK1, respectively. The 61-kD band contained sequence V and was derived from both PCK1 and PCK2. The differences in the N termini present in the PCK mixture versus the isolated 62- and 61-kD bands may have been due to the increased ability to detect the amino acids released from low-abundance polypeptides in the purified bands compared with the mixture containing several different N termini. Alternatively, there may have been variations between the two enzyme preparations used in the analyses.

	DISCUSSION

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The U. panicoides PCK Multigene Family

The sequencing of cDNAs spanning the entire PCK2 ORF has shown that the PCK2 subunit is 96% identical to PCK1. None of the differences are likely to affect the active site of the ATP-dependent PCK (Matte et al., 1996). The concentration of sequence differences at the N and C termini has allowed us to show that both proteins are expressed in U. panicoides leaves. In fact, all four PCK genes examined here may yield a protein product. RT-PCR analysis indicated that both PCK3 and PCK4 are also transcriptionally active, but the possibility that one or both is translationally silent has not been ruled out.

The finding of two members of a gene family in close proximity to one another and in the same transcriptional orientation is not unknown in plants. Similar gene arrangements have been observed for the alternative oxidase genes AOX1a and AOX1b (Saisho et al., 1997) and the drought-induced genes rd29A/rd29B (Yamaguchi-Shinozaki and Shinozaki, 1993) in Arabidopsis, and the catalase genes cat1 and cat2 in castor bean (Suzuki et al., 1994).

Control of PCK Gene Expression

Role of PCK Subunits

Regulation of PCK in Plants

The second type of plant PCK is slightly smaller, 67 to 70 kD, and is not subject to phosphorylation. This enzyme type has only been found in the leaves of some PCK-type C₄ grasses, including U. panicoides (Walker and Leegood, 1996; Walker et al., 1997). So far, only the predominant PCK found in leaf tissues, and therefore involved in photosynthetic carbon assimilation, has been examined in these species. It will be interesting to see whether the enzyme composed of PCK3 and/or PCK4 subunits is regulated differently than the photosynthetic enzyme.

	FOOTNOTES

   Received December 28, 1998; accepted April 27, 1999.

	ABBREVIATIONS

Abbreviations: PCK, PEP carboxykinase. RT-PCR, reverse transcription-PCR.

	LITERATURE  CITED

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