molecular characterization of a phosphoenolpyruvate carboxylase in the gymnosperm picea abies...

Plant Molecular Biology 32: 923-936, 1996. (~) 1996 Kluwer Academic Publishers. Printed in Belgium.

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Molecular characterization of a phosphoenolpyruvate carboxylase in the gymnosperm Picea abies (Norway spruce)

Manfred Relle and Aloysius Wild* Institut fiir Allgemeine Botanik der Johannes Gutenberg- Universitiit Mainz, D-55099 Mainz, Germany (* author for correspondence)

Received 18 March 1996; accepted in revised form 14 August 1996

Key words: PEPC, C3 metabolism, gene expression, evolution, gymnosperm, Picea abies

Abstract

Phosphoenolpyruvate carboxylase (PEPC) genes and cDNA sequences have so far been isolated from a broad range of angiosperm but not from gymnosperm species. We constructed a cDNA library from seedlings of Norway spruce (Picea abies) and identified cDNAs coding for PEPC. A full-length PEPC cDNA was sequenced. It consists of 3522 nucleotides and has an open reading frame (ORF) that encodes a polypeptide (963 amino acids) with a molecular mass of 109 551. The deduced amino acid sequence revealed a higher similarity to the C3-form PEPC of angiosperm species (86-88%) than to the CAM and C4 forms (76-84%). The putative motif (Lys/Arg-X-X-Ser) for serine kinase, which is conserved in all angiosperm PEPCs analysed so far, is also present in this gymnosperm sequence. Southern blot analysis of spruce genomic DNA under low-stringency conditions using the PEPC cDNA as a hybridization probe showed a complex hybridization pattern, indicating the presence of additional PEPC-related sequences in the genome of the spruce. In contrast, the probe hybridized to only a few bands under high-stringency conditions. Whereas this PEPC gene is highly expressed in roots of seedlings, a low-level expression can be detected in cotyledons and adult needles. A molecular phyiogeny of plant PEPC including the spruce PEPC sequence revealed that the spruce PEPC sequence is clustered with monocot and dicot C3- form PEPCs including the only dicot C4 form characterized so far.

Introduction

The cytosolic phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) catalyses the /%carboxylation of phosphoenolpyruvate (PEP) utilizing HCO3- to yield oxaloacetic acid and inorganic phosphate. The enzyme, which had been demonstrated for the first time in 1953 by Bandurski and Greiner in leaves of spinach [3], is only found in plants and microorganisms; it is missing in animal tissue and in fungi. The most extensively studied function of the enzyme is the initial fixation of atmospheric CO2 in C4 and CAM plants. In C3 plants the enzyme fulfils different functions [34], one of them being the replenishment of the citric acid cycle, a function it also carries out in microorganisms. A clearer

The nucleotide sequence data reported will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number X79090.

defined function of the enzyme in C3 plants was only found in legume root nodules. There PEPC provides, on the one hand, the carbon skeletons for the fixation of nitrogen and, on the other, malate and succinate, which come from secondary reactions of PEPC [27, 50].

In C4 and CAM plants the enzyme is encoded in a small, well characterized gene family, whose isozymes carry out different functions in metabolism [ 12, 21, 22, 36]. Since the PEPCs in C3 plants have not been studied as much as those in C4 plants, only a few PEPCs of C3 plants have been characterized on the molecular level [28, 44, 48, 56]. Although there are hints that PEPC in C3 plants may also be coded for by several genes [43, 48, 54], isoforms from C3 species have not yet been isolated.

The conifer Norway spruce (Picea abies L. Karst.) is one of the most important species of European trees.

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As a relatively undemanding but productive lumber tree the spruce was introduced into areas outside its natural range, such as the boreal coniferous forests and the high-lying regions of central Europe. Despite its enormous ecological and economic importance very few studies of the molecular biology of Norway spruce have been carried out so far [4, 10, 20, 33, 57].

To study PEPC in a gymnosperm C3 plant our first step was to clone, sequence and analyse a PEPC encoding cDNA from Norway spruce. Using the PEPC cDNA as a hybridization probe we then demonstrated the high level of expression of PEPC mRNA in roots of seedlings and the presence of other PEPC-related sequences in the genome. In addition, we examine and discuss the phylogenetic relationships between the PEPCs in different prokaryotic and plant species in an attempt to get insight into the evolution of this enzyme.

Materials and methods

Plant material

Spruce (Picea abies L. Karst.) seeds were obtained from the Staatliche Samenklenge Elmstein (Elmstein, Germany). The seeds were germinated and grown on moist vermiculite in a growth chamber at 25 °C for 2-3 weeks in white light (16 h light, 8 h dark). The seedlings were then cut into three pieces (cotyledons, stem, root) and stored at -80 °C until use in the isolation of RNA or DNA from cotyledons and roots.

Preparation of total RNA and poly (A) + RNA

RNA was extracted from 2-3-week-old seedlings (roots and cotyledons) and from needles of mature trees. The plant material was ground with a mortar and pestle under liquid nitrogen. The resulting powder was used to isolate RNA as described in a previous paper [49]. Poly(A)-enriched RNA was obtained from total RNA by a single passage through an oligo (dT)-cellulose spin column (Pharmacia LKB, mRNA purification kit). Poly(A) + RNA was obtained from total RNA by use of magnetic monosized microspheres (Dynabeads Oligo dT) according to the manufacturer's instructions (Dynal).

Reverse transcription and polymerase chain reactions (RT-PCR)

For the screening of the cDNA library we used a homologous PEPC-specific probe generated by RT-PCR. To eliminate contamination of the RNA with genomic DNA 50 #g of total RNA from 2-year-old needles were treated with 40 units of DNase I [52]. 10 #g of this RNA was then used to synthesize single-stranded PEPC-cDNA using AMV reverse transcriptase (AGS, Heidelberg, Germany) and 50 pmol of the 3' PCR primer (ppc primer 4, see below). After denaturing the reverse transcriptase at 95 °C for 5 min, 10 #1 of first- strand reaction mix was used directly for PCR with Taq DNA polymerase (Boehringer, Mannheim, Germany) in a total volume of 100 #1. DNA fragments were amplified in a programmable thermocycler (Hybaid, Middlesex, UK) with degenerate primers designed on the basis of published PEPC gene and cDNA data (see Fig. 1).: ppc 1: 5'-GTI TTI ACI GCI CA(T/C) CCI AC-3' ppc 4: 5'-CCI CCI CGI CC(G/A) TG(G/A) AA-3' Amplifications were carried out in 32 cycles of 92 °C for I min, 45 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min with a 5 min extension of the 92 °C step in the first cycle and a 5 min extension of the 72 °C final step in the last cycle. The resulting amplified products (ca. 1.4 kb) were separated on a 1% agarose gel and extracted via DEAE cellulose membrane binding and elution according to standard procedures [52]. The purified products were then partially sequenced by a modified chain termination method [2].

Construction and screening of the cDNA library

The cDNA library was constructed from 5 #g of poly(A) enriched RNA isolated from cotyledons. cDNA cloning was carried out with the TimeSaver cDNA Synthesis Kit and the Directional Cloning Toolbox according to the manufacturer's instructions (Pharmacia LKB, Uppsala, Sweden). The double- stranded cDNA (with EcoRI and NotI ends) was size- fractionated (>400 bp) by passage through a Sephac- ryl S-400 column and ligated to EcoRI and NotI- digested dephosphorylated phage A ExCell arms (Phar- macia LKB, Uppsala, Sweden). Packaging was performed with the Gigapack system (Stratagene, La Jolla, CA). The resultant recombinant phages were amplified before screening by infection of the Escherichia coli host strain XL1-Blue MRF'. Putative PEPC cDNAs were detected by plaque hybridization. Phage plaques

1 GCTTGTCCT~C~TAGAAACATGGCTTTGAGCATCTCAACCTG~TOTTGAAAT~CTTCCGAGCCCGAGA M A L S I S T W I L K s T O ~

73 TCATTGAAACTTTG~GAAATCGTGTTGTTG~TTTTTATTGTTGCAGAGGAAAGATCAG~TTTTGAGATT

145 TTGGCG~GCGG~CTTGGATTTAG~GAAATCTGTTG~TC~CGCGC~C~CTTGGAGAA~TG~

M A R N N L E K M A I 0

217 ATCCATTGATGCCCAGATGAGGCTCTTGGTTCCTGGAAAAGTTTCTGAGGATGAC~GTTGATTGAGTACGA S I D A Q M R L L V P G K V S E D D K L I E Y D 3 4

289 TGCCCTTCTACTGGATCGCTTCCTTGACATCCTGCAGGACTTACATGGAG~TATCAGGGCGATGGTTCA A L L L D R F L D I L Q D L H G E D I R A M V Q 5 8

361 AG~TGCTATGAGCGTTCTGGTG~TATGAGGGAAAG~TGATCCTCAC~GCTGG~GAGTTGGGAAATGT E C Y E R S G E Y E G K N D P H K L E E L G N V 8 2

433 ATT~C~GTCTG~TCCTGGG~TTC~TTGTTGTGGCCAGCTCATTTTCCCACATGCTT~TTTAGCT~ L T S L D P G D S I V V A S S F S H M L N L A N I 0 6

505 TTTAGCTG~G~GTTCAGATTGCTTACCGGCGCCGAAATAAAATAAAGAGGGGCGGCTTTGCAGATGAAAG L A E E V Q I A Y R R R N K I K R G G F A D E S I 3 0

577 C~TGC~CTACTG~TCAGACATTG~GAAACTTTTAAAAGGCTTGTAAACCAGTTAGGAAAATCACCGGC N A T T E S D I E E T F K R L V N Q L G K S P A I 5 4

P

649 AGAGGTGTTTGATGCTCTT~G~T~GACTGTTGATTTGGTGTTGACAGCACATCC~CACAGTCAGTCCG E V F D A L K N Q T V D L V L T A H P T Q S V R I 7 8

721 CAGATCATTGCTG~G~GCATGCTAGGATTCGG~TTGCTTGTCTCAGTTATATGGGAAAGATATTACCCC R S L L Q K H A R I R N C L S Q L Y G K D I T P 2 0 2

793 TGATGAG~G~GGAGCTTGATG~GCTTTGCT~GAGATTC~GCTGCCTTCCGCACAGATGAAATTCG D E K Q E L D E A L L R E I Q A A F R T D E I R 2 2 6

865 GCGTACTCCTCCTACTCCAC~GATGA~TGCGAGCAGGAATGAGCTATTTTCATGAAAC~TTTGGAAGGG R T P P T P Q D E M R A G M S Y F H E T I W K G 2 5 0

937 TGTCCCT~GTTTTTACGCCGTATTGATACTGCCCTG~GTCAATTGGGATC~TG~CGAGTGCCGTAT~ V P K F L R R I D T A L K S I G I N E R V P Y N 2 7 4

1009 TGCACCTCTTATA~GTTTTCTT~TG~TGG~GGT~TC~TGGA~TCCTAGGGT~CTCCAG~GT A P L I Q F S S W M G G D R D G N P R V T P E V 2 9 8

1081 ~C~GATGTATGCTTACTTGC~G~TGATGGCAGCAAATTTATATTATTCCCAGATAGAGGATCTTAT T R D V C L L A R M M A A N L Y Y S Q I E D L M 3 2 2

1153 GTTT~GTTGTCCATGTGGCGTTGTAGT~T~GCTGAGAG~C~GCCCTAC~CTCCATAGTGCATCAAA F E L S M W R C S D E L R A R A L Q L H S A S K 3 4 6

1225 GAAA~TGCA~GCATTA~TAGAATTTTGGAAA~GATTCCTCCAAATGAACCTTTTAGGGTGATATTGGG K D A K H Y I E F W K Q I P P N E P F R V I L G 3 7 0

1297 A~TGTAC~GATAAATTGTAC~TACTC~GAACGTACTCGCC~TTACTTTCT~TGG~TTTCTGACAT D V R D K L Y N T R E R T R Q L L S N G I S D I 3 9 4

1369 ACCA~GG~GT~CCT~ACAAATATT~C~GTTTTTG~GCCACTTG~CTTTGTTACCGATCACTGTG P E E V T F T N I D E F L E P L E L C Y R S L C 4 1 8

1441 TTCTACTGGGGACCAGCC~TTGCAGATGGCAGTCTTCTT~TTTTATGCGTC~GTTTC~CATTTGGTTT S T G D Q P I A D G S L L D F M R Q V S T F G L 4 4 2

1513 GTCATTTGTT~GCTGGATATTA~CAGG~TCUGACAGACACAGTGATGTTGCTGATGC~TTAC~GGCA S F V K L D I R Q E S D R H S D V A D A I T R H 4 6 6

1585 TTTAGGCATTGGATCCTACAAAGAGTGGTCA~GG~C~CGAC~GCATGGCTCTTGTCAG~CTGC~GG L G I G S Y K E W S E E Q R Q A W L L S E L Q G 4 9 0

1657 GAAACGTCCCTTGTTTGGGCCGGACCTCCCAAAGACA~TGAGGTTCGAGATGTTCTAGACACATTTCATGT K R P L F G F D L P K T D E V R D V L D T F H V 5 1 4

1729 ~TATCTGAGCTTCCTGCTGAC~TTTTGGAGCTTATATTATTTC~TGGC~CTGCAGCATCAGATGTTTT I S E L P A D N F G A Y I I S M A T A A S D V L 5 3 8

1801 AGTGGTTGAGTTATTACAACGGG~TGCCATGTGAAA~CCATTACGTGTTGTACCATTATTTGAG~GCT V V E L L Q R E C H V K K P L R V V P L F E K L 5 6 2

1873 TGCAGATCTAGAGGCTGCTCCTGCTGCTTTGGCTAGATTGTTTTC~TAAACTGGTACAGAAACAG~TTGA A D L E A A P A A L A R L F S I N W Y R N R I D 5 8 6

1945 TGGAAAGC~G~GTCATGATTGGTTACTCTGATTCTGGAAAGGATGCTGGTAGGTTGAGTGCAGGTTGGGC G K Q E V M I G Y S D S G K D A G R L S A G W A 6 1 0

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Figure 1.

926

4 2017 TTTGTACAAAGCACAGG~GACCTTATAAAGGTTGCTAAAG~TTTGGTATT~GTTGAC~TGTTCCATGG

L Y K A Q E D L I K V A K E F G I K L T M F H G 6 3 4

2089 TCGTGGAGG~CTGTAGGCAGGGGGGGAGGCCC~CCCATCTGGCCATATTGTCAC~CCTCCAGACAC~T R G G T V G R G G G P T H L A I L S Q P P D T I 6 5 8

2161 TCATGGCTCTTTTCGGGTCACTGTTC~GGGG~GTCATAG~CAGTCATTTGGTGAGG~CATTTGTGTTT H G S F R V T V Q G E V I E Q S F G E E H L C F 6 8 2

2233 CAGAACTCTCC~CGATTTACTGCTGCCACTCTTGAGCATGG~TGCGGCCTCCAGTTGCACCAAAGCCTGA R T L Q R F T A A T L E H G M R P P V A P K P E 7 0 6

2305 GTGGCGTGAACTGATG~TGAAATGGCTGTTGTTGCTACG~G~GTACAGGTC~TTGTTTTCCAGGACCC W R E L M D E M A V V A T K E Y R S I V F Q D P 7 3 0

2377 ~GATTCGTTG~TATTTCCGTTCTGCAA~C~G~TTGGAGTATGGTCG~TG~CATCGGGAGTCGCCC R F V E Y F R S A T P E L E Y G R M N I G S R P ~ 7 5 4

2449 CTCAAAAAGG~GCC~GTGGGGGCATTGAGTCACTCCGTGCCATCCCATGGATATTTGCTTGGACACAAAC S K R K P S G G I E S L R A I P W I F A W T Q T 7 7 8

2521 TCGATTTCATCTTCCTGTTTGGCTTG~TTTGGTG~GCATTC~GCATGTTATGGAGAAG~TAT~GAAA R F H L P V W L G F G A A F K H V M E K D I R N 8 0 2

2593 TCTCCATATGCTGCAGCAGATGTAC~TG~TGGC~TTCTTTCGGGTTAC~TTGATCT~TTGAAATGGT L H M L Q Q M Y N E W P F F R V T I D L I E M V 8 2 6

2665 TTTTGCAAAAGGT~TCCAGGGATAGCTGCTTTGTAT~CAAACTACTGGTATCTGATGATTTGTGGGCCAT F A K G D P G I A A L Y D K L L V S D D L W A I 8 5 0

2737 TGGTGAAAAATTGAGAGCT~CTATGGTGAAACCAAAGATTTGCTACTGCAGGTTGCTG~TAAAGATCT G E K L R A N Y G E T K D L L L Q V A G H K D L 8 7 4

2809 ACTTG~GGTGATCCATACTTGAAACAGCGACTCAGACTTCGTGACTCATACATTAC~CTCTGAATGTTTG L E G D P Y L K Q R L R L R D S Y I T T L N V C 8 9 8

2881 TCAGGCTTATACTTTGAAAAGAATTA~GACCCC~CTATCATGTT~TCTTAGGCCTCATTTGTCG~GGA Q A Y T L K R I R D P N Y H V N L R P H L S K E 9 2 2

2953 ~GTTC~CCAAACCGG~GCTG~TTGGTCAAACTG~TCCAAC~GTGAGTATGCACCAGGTCTGGAGGA S S T K P A A E L V K L N P T S E Y A P G L E D 9 4 6

3025 TACATTGATTCT~CCATG~GGGCATAGCAGCTGGTATGCAG~CACTGGTTAG~GGATTTTGGAAACGG T L I L T M K G I A A G M Q N T G S T O P 963

3097 ATCAGGCATTTTCTCATGTTAGGAGTCAG~CTAT~CGG~GTTTCTTGAGACCTACTTCTGAGGGACCTT 3169 CTTCAGATTCCAGTATTT~GGCAGCTTCTAAATATGTCAAAGCACCAGCAAG~TGATCTGC~GTGTA~ 3241 TATGGTCATTT~T~GCTC~G~GAGAGCAAATGTTGGTTGTCAGTTGACAACTTTCAAG~TTTTATTA 3313 T~CGAGTCTAGGATCATATATGATCTTAGGATCCCT~TATATATGAAAAGCCCTAAAACATGTAGTTTTG 3385 TG~CTGGACTT~GGACCAGGACTCCTTTATATGC~GGCTTGGG~GTAC~TGATTT~CTTGATG~T 3457 G C A T T ~ T ~ T G A T C T T T A G C A T G C T G T T G T T T G T G G ~

Figure 1. The nucleotide sequence of PAPPC 14/2 with the predicted translation products indicated below the coding sequence. The amino acids are given in single-letter code. The phosphorylation motif KMAS and the putative polyadenylation signal are underlined. The terminator codons of the two ORFs and the poly(A) tail are shown in bold. The sequence context around the potential initiator codons of the ORF coding for PEPC is doubled-underlined and shown in bold. Arrows indicate the two primer sites used in RT-PCR in generating the probe PEPC-1409 for screening the spruce cDNA library. The site for primer ppc4 with a sequence complementary to that shown in Fig 2 is indicated by the arrow pointing towards the left.

were transferred to Hybond-N+ membranes (Amer- sham, Buckinghamshire, UK) and the filters were prehybridized for 1 h. Hybridization was performed overnight at 60 °C in 5 × SSC, 0.5% SDS, 5 x Den- hardt's solution and the labelled probe. The probe was the amplification product obtained with the primer pair ppc 1 and ppc4. It was [a32p]-dATP labelled using random primers [17] according to the protocol of the Mega Prime DNA-Labeling Kit (Amersham, Buck- inghamshire, UK). The filters were then washed under low-stringency conditions for a few minutes at room

temperature with 2 x SSC, 0.2% SDS to remove the bulk of the radioactivity and then for 30 min at 60 °C with 2 x SSC, 0.2% SDS and for 30 min at 60 °C with 1 x SSC, 0.1% SDS, with one change of solution. The blots were then exposed to Kodak X-ray film (Kodak, Rochester, NY) for autoradiography at - 8 0 °C with intensifying screens. After three rounds of screening five positive clones out of 80 000 phages were plaque purified and used to infect E. coli strain NP66. This special strain of E. coli enables the in vivo release of

recombinant pExCell plasmid according to the manufacturer's protocol.

Subcloning and DNA sequencing

All 5 clones were partially sequenced from both ends. One isolated full-length clone (3.5 kb) called pPAP- PC14/2 was selected for complete sequencing and sequence evaluation. The isolation of the recombinant plasmids was carried out by alkaline lysis of the bacteria [5]. DNA preparations of up to 20 #g were carried out with the Qiaprep-spin Kit and preparations of up to 100 #g were carried out with the Qiagen- Midi-Kit (both from Qiagen, Diisseldorf, Germany). Since the yield of recombinant plasmid-DNA isolated from the E. coli strain NP66 was small, the E. coli strain JM 109 was transformed with the isolated plasmids. Dideoxy sequencing of both strands was carried out using a thermal cycle amplification system (PRISM Ready Terminator Cycle Sequencing Kit, Applied Biosys- terns, USA) with fluorescent-labelled nucleotides. The automated sequencing (Applied Biosystems, 373A, USA) was carried out in the Institut fiir Molekulargen- etik, gentechnologische Sicherheitsforschung und Ber- atung (Professor E.R. Schmidt, Universit~it Mainz). The M13 Universal and M13 Reverse Sequencing primers (US Biochemicals, Cleveland, OH) were routinely used for sequencing of the subclones. The sequence of the first strand was obtained from deletion subclones generated with Exonuclease III (double- stranded Nested Deletion Kit, Pharmacia, Uppsala, Sweden). Later plasmids were obtained by subcloning of restriction fragments and used to determine the complete sequence.

Sequence and phylogenetic analyses

Computing was carried out on a PC microcomputer using the PCGENE (IntelliGenetics, Mountain View, CA) and the MEGA [32] software packages. Stand- ard sequence compilation was performed with the pro- grams PCOMPARE and PALIGN of the PCGENE software package. Multiple sequence alignments were performed with the program CLUSTAL of the PCGENE package and later with the CLUSTAL W program [59]. For the phylogenetic analyses the MEGA package was chosen. The neighbour-joining (NJ) method used for 'tree' construction is based on distance matrix analysis and allows for comparison of the evolutionary rate of each protein [51 ]. Bootstrap analysis with 100 replications was completed to determine the confidence level

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of branches within the NJ tree [18]. Molecular mass and theoretical isoelectric point prediction was carried out with the PROSIS package (Hitachi).

Northern blot analysis

1 /zg of poly(A) + RNA was denatured and fractionated on a 1% agarose, 2.2 M formaldehyde gel [52]. The RNA was transferred onto a positively charged Hybond-N+ nylon membrane (Amersham) in 20x SSC according to the procedure described by Chomczynski [9]. The full-length PEPC cDNA clone PAPPC 1412 and the rbcS PCR product (positive control) were labelled by the random primer method with 32P-dATP using the Mega Prime DNA-Labeling Kit (Amersham). Hybridization was carried out in com- mercially available hybridization solution (Stratagene) plus the labelled probes at 60 *C overnight. After hybridization the membranes were washed under low- stringency conditions with 1 x SSC, 0.1% SDS for 15 min at room temperature and twice with I x SSC, 0.1% SDS for 15 min at 60 ° C. The filter were then subjected to autoradiography for 3 days a t -80 °C with intensifying screens. After this the same blot was washed under high stringency conditions with 0.1 x SSC, 0.1% SDS at 65 °C and once more exposed to film for 7 days.

Southern blot analysis

For Southern blot hybridization total genomic DNA was isolated from frozen cotyledons of 2-3-week old seedlings employing a two-step procedure. First, total DNA was prepared by a modification of the CTAB method of Murray and Thompson [45]. The cotyledons (up to 5 g, fresh weight) were ground to a fine powder with a mortar and pestle. The powdered needles were added directly to 50 ml of extraction buffer (200 mM Tris pH 7.5, 700 mM NaC1, 10 mM EDTA, 15 mM DTT, 2% TNS) and incubated at 60 °C for 30 rain. After the addition of 10 ml of 10% (w/v) cetyltrimethylammonium bromide (CTAB)/0.7 M NaC1 the suspension was incubated at 60 °C for additional 15 rain and subsequently centrifuged (10 min, 3800 × g) to remove the debris. The solution was extracted twice with chloroform/isoamyl alcohol (24:1), brought to a final concentration of 10 #g/ml RNase A and incubated at 37 °C for 1 h. In a second step the crude DNA solution was loaded on an anion- exchange column (Qiagen Genomic-tips 500/G) which was equilibrated with TNE buffer (200 mM Tris pH 7.5, 700 mM NaCI, 10 mM EDTA, 0.15% Triton X-

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100). The remainder of the procedure followed the handbook of the manufacturer. 5 #g of high-molecular- weight DNA was cleaved with the AsnI, DraI and SspI restriction enzymes and subjected to electrophores- is in a 0.8% agarose, 0.5 x TBE gel. The separated DNA was transferred to a Hybond-N+ nylon membrane (Amersham) in 0.4 M NaOH, 1 M NaC1 under denaturing conditions. Hybridization of the Southern blot was performed as described above for the northern blot filters with the exception that a 1.0 kb SspI fragment of the cDNA clone PAPPC14/2 was used as a probe.

Standard techniques of molecular biology

These were carried out according to published proto- cols [52].

Results

Cloning and analysis of the spruce PEPC cDNA clones

A spruce cDNA library was constructed from poly(A) + RNA extracted from cotyledons of 3-week-old spruce seedlings. Because of the evolutionary distance between angiosperms and gymnosperms a homologous spruce PEPC cDNA fragment was synthesized, using the RT-PCR technique in order to screen the cDNA library. For the amplification reactions two degenerate oligonucleotides were designed with sequences complementary to highly conserved regions of published PEPC sequence data. The two primers corresponding to amino acids 168-174 and 632-637 of the spruce PEPC (Fig. 1) and total spruce single strand cDNA were used in PCR reactions which resulted in two DNA fragments. The shorter one was 1409 bp long (PEPC- 1409) and turned out to be homologous to published PEPC gene and cDNA sequences. To get a full- length clone for PEPC, labelled PEPC-1409 was used to screen the cDNA library.

Out of 80 000 recombinant phages screened, five positive clones with insert sizes of ca. 2.8 kb (3 clones) and ca. 3.5 kb (2 clones) were obtained and partially sequenced from both ends. All five clones were identical in overlapping regions, indicating that the five cDNAs isolated are coded for by a single gene. Whereas both of the longer clones encode the complete PEPC, the shorter clones encode a PEPC lack- ing the amino terminus of the enzyme. The 3' trailing

sequence is equally long in four of the five clones, whereas one of the 2.8 kb long clones has a longer 3' region. This variation could be explained by the altern- ative usage of one of two polyadenylation signals (data not shown). One of the equally long full-length clones, called pPAPPC14/2 (Fig. 1), was selected for further sequence evaluation. The 3522 bp long insert has a major open reading frame (ORF) of 2889 bp, 187 bp of a 5' leader sequence, and a 3'-untranslated region of 446 bp (including a poly(A) tail of 28 bp). The ORF codes for a polypeptide of 963 amino acid residues with a predicted molecular mass of 109 551 Da and a theoretical isoelectric point of 5.9. The 5' leader contains another short open reading frame encoding an 11 aa peptide. Two ATG triplets (nucleotides 188-190 and 212-214) are possible translation initiation sites in the PEPC encoding ORE because both methionine codons are in an optimal context for translation initiation [7]. A putative polyadenylation signal is found at the 3' end (positions 3462 to 3466) of the PAPPC14/2 cDNA.

Protein structure

The comparison of the putative protein sequence of PAPPC1412 with published plant PEPCs (Table 1) revealed a high degree of similarity (86-88%) to C3- form PEPCs. A reduced level of similarity exists to the C4 isoforms of Sorghum and maize PEPCs (76% and 79%), to the salt-induced CAM-form of ice plant (M. crystallinum) PEPC, and to the C4 form of the dicot species Flaveria trinervia (82% and 84%).

The spruce PEPC contains the putative phosphorylation motif for serine kinase [24] (Fig. 2) which is present in all angiosperm PEPC sequences described so far. Other sequence motifs proposed to be essential for the oxaloacetate formation (VLTAHPT, 168-174), the active site (GYSDSGKDAG, 594-603), and the substrate-binding site (FHGRGGTVGRGGGP, 632- 645) in all PEPC sequences, are also present in the spruce sequence. Cysteine residues may be involved in subunit interaction and the redox regulation [8]. Of the seven cysteine residues that are conserved in the PEP-carboxylases of plants only residue 420 is sub- stituted by the amino acid threonine. Besides other preserved areas of unknown function, the conservation of the carboxy terminus with regard to length as well as sequence should be noticed. The C-terminal sequence motif LTMKGIAAGMQNTG exists slightly modified in the carboxy-terminus of all plant PEPCs (Fig. 1) and the PEPCs of Anabena variabilis, Anacystis nidulans, Corynebacterium glutamicum, and Escherichia

929

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~ I .............................................. MST- -VKLD RLTS I DAQLRLLAPKKVSE DDKLI EYDALLLDR FLDI LQNLHGEDI KETVQELYE 62 ~ C 4 .............................................. MAS ..... E RHH S I DAQLRALApGKVSEE --LIQY DALLVDR FLDI LQDLHGPSLRE FVQECY£ ~7

E . ( ~ I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MNEQy SAL R S NVSMLGKVLGE T I KDALGEHI LERVETI R ~ C . g ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MTDFLRDDI R FLGQI LGEVIAEQEGQEVYELVEQAR

MLAVS LR DHG FP SATVQI R PAFCVQS DVVG SGN P PRMN Y CQNARTAMSAALQS S D DAFRTVSS PLAT DL DLS S PLE F FL RH R LTVVE E L~EVVLRQECGQE LV DI LTQLR 110

~J4f ' tJ~O 1 4 ~ l Q ~ Y I ~ m ~ 3 ~ f G . D I ~ D 8 1 " ~ ' ~ " A S I I F ~ . . . . . . . . . . . . . . . . . . . . . . . . . QZA'dI~z,~-TIK]R~O~ADII, dR4~'I'I~IHDZI~CI~IK~V l U ~l~e ~.~ VAAEYETKHDLQKLDELGKMI TSLDPGDS I VIAKSLSHMLNLANLAEEV ......................... QIAYRRRI K- LKKGDFADZNSAITESDIEETLKRLV ~4~ TO~ICK~ L SAE Y EGKH DPKKLE E LGNVLT SL DPGDS IVIAKAFSHMLN LAN LAE EV ......................... QIAYRRRQK-LKRGDFADENNATTESDIEET FKKLV 146

FkWMJO C4 LSAEYEGKHDPKKLDELGSLLTSLDPGDSIVIAKAFSHMLNLANLAEEV ......................... QIAYRRRI K-LKSGDFADEANATTESDI EETFKRLV ~46

p~Nt I QSAEY ERTHDPKKLEELGSMVTSLDAGDSI WAKS FSHMLNLANLAEEV ......................... QI SRRKRVKKVKKGDFMDENTAMTESDMEETLRRLI 14T ~U111C4 VSADY £GKKDT SKLGE LGAKLTGLAPADAI LVASS I LHMLN LAN LAE EV ......................... ELAHRRRNSKLKHGDFSDEGSATTESDI EETLKRLV ~42

I:.CO~ KLS KS SRAGN DAN RQE LLTTLQNLSN DE LL PVARAFSQ FLN LANTAEQy ..................................... HS I S PKGEAASN PEVIARTLRKLK 11~

C . O I W ~ -LTS FDIAKGNA£MDSLVQVFDGI T PAKATPIARAFSH FALLANLAE DLYDEELR EQALDAG DT pP ................................. DSTLDATWLKL 11Z

A . I l l d u l i l s DLTSPEGQAPEVGGEALVQVI ETLELSDAI RAARAFALY FQLINIVEQHYEQTQy QLAYERSRLE PLPGPDESPEGLHT I EI pQHQLDPFAAVI PLNQDPAT FQTLFPRL

E~:cdl 8cqhum C4 Ice l int 1 - - C 4 Tobacco Spruce Maize C.1 i ~ ~ ~ ~ i i ~ ~ ~ i ~ i i i i ! ~ ~ ~ ~ TM 2t6 245 261 260 2M 212

C . ~ NEGNVGAEAVADVLRNAEVAPVLTAH PTE T RR RTV FDAQKWI TTHMRE RHALQSA E PTARTQ S KL DE I E KN I RRR I T I LWQTALI RVAR pR I E DE I EVGLRYY KLS LLE E 222

A . ~ RQLNVPPQMIQELT DRLDI RLVFTAH PTE IVRHT I RDKQRRIAYLLRQLDELETG KN RGFRELEAQNI RQQLTZEI RLWWRT DELHQFKPTVLDEVDYALHY FQEVL FEA 3~ # ##### # # # # ##

VI~Gm-A~I~tDTAZ,K8 Z ~ T Q]r ~ ~ A]R~AMZ, I~BQT ~ ] I Z J R A P J k T ~ Q ~ , B J i A - - m o ~ l r Z~]qEKQ Maize C3 VPKFLRRVDTA~KN IS~ NERVPYNAPLIQFSSWMGGDRDGNPRVTpEVTRDVCLL ~ N L Y C S Q ~ £DLMFELm4WRCS DEL~M~ADVLHLS--TKKDAKHy~ £~T~K 380 T O ~ VPKFLRRVDTALKN IGI NERLPYNAPL IQFSSWMGGDRDGNPRVTLEVTRDVCLL ARMMAANLYYSQI EELMFELSMWRCN DDLRI RAAELYRS -- SRRDTKHYI E FWKT 368

~'~IV~H~ C4 VPK FLRRVDTALKN I G I NE R FPYNAPL I Q FS S%~4GGDR DGN pRVT PEVTR DVCLL ~ T SNMY FSQI E DLMI EMSMWRCN SE LRVRAE ELy RT - -ARKDV}CMy I E FWKQ Me k:e ~ 1 ~ 1 VPKFLRRLDTALKNIGITERVPYNAPL IQFSS~GGDRDGNPRVT PEVTRDVCLL AR~MY FSQI DELMFELSMWRCTDELRERAEELHKY - -S}¢RDSKHYI E F~Q 380

~ C 4 VPKFLRRVDTALKN I GIN E R LPY DVPL I K FCSWMGG DR DGN PRVT PEVTR DVCLL S RF~AAN LY I N QVE D124 FELSMWRCN DE LRARAEEVQS T PASKKVTKYy I E FWKQ Me

E.~ VPNY LRE LN EQLE EN -LGY KL PVE FVPVR FT SWMGGDR DGN PNVTADI TRHVLL L S RWKAT DL FLKD I QVLVS ELSMVEAT pE LLALVGE .................... ~0~

C.~IK I PR I NRDVAVE LRE R - FGEGVPLK- PVVKPG SWI GGDH DGN PYVTAETVE y ST H R AAETVLKYYARQLH S LE H ELSLS DRMNKVT pQL LALADAGHN DVP .......... ~0

I PLLYQR FRLALQGT- FPDLQP PRYN FCQFGSWVGSDRDGNPSVTSAVTWQTACY QRSLVLDRYI TAVEHLRNVLSLSMHWSEVLPELLSSLEQ- -ESMLFPETYEQLAV 437 # # # ## # ~ #### ## # #

Z IPIP~]UPIN~LV~ . T . ~ __ ~Z ~D ~ I~I~'~e~TTD]IFZJ~I~LaI~C TJR~-~-J~TG~p TADG" - - - B L L D I q ~ B ~ ] P G / , ~ I ~ K ~ D Z I ~ T 464 M&~ze C~ VPPNEPYRVI LSDVRDKLYNTRERSRELLSSGHS DI PEEATLTNVZQLLE PLELC YRS LCACGDSVIADG . . . . TLL D FLRQVST FGLS LVRLDI RQE S DRHT DVL DAI T 4~

T ~ ' . ~ - O I PPSEPYRVI LGDVRDKLYQTRERTRQMLAHGI S DI PE DATYNNVEQFLE PLELC YRSLCECGDRPIADG .... SLLDFLRQVST FGLS FVRLDI RQESDRHTDVLDAI T 464 ~l~v~ C4 I PPNQPYRVI LGDVRDK'LYNTRERSRHLLVDGKS DI PDEAVYTNVEQLLE PLELC YRSLCDSGDHVIADG .... SLL DFLRQVST FGLSLVKLDI RQESDRHTEVLDAI T 464

k:e ~11~ I I PSSEPYRVI LADVRDKLYYTRERSRQLLASEVSEI PVEAT FTE I DQFLEFLELC y RSLCACGDRPVADG .... SLL D FMRQVAT FGLCLVKLDI RQE SE RHT DVMDAI T 466

~W11C4 I PPNEPYRVI LGAVRDK'LYNT RERARHLLATGFSEI SE DAVFTKI EEFLE PLELC YKSLCECGDKAIADG .... SLLDLLRQVFT FGLSLVKLDIRQESERQT DVI DAI T 461 S . ~ | EGAAE PY RY LM~N LRSRLMAT QAWLEARLKGE E L p Kp- EGLLTQNE E LWE PLYAC yQS LQACGMGI IAN G .... DLLDTLRRVKCFGVPLVRI DIRQESTRHTEALGELT 410 C ~ S RVDE PY RRAVHGVRGR I LATTAE LI GE DAVEGVWFKVFT pyAS pE E FLN DALT I DHS LRE SK DVL I ADD .... RLSVLI SAI ES FGFNLYALDLRQNSESYE DVLTEL F 4~$ A . ~ RYRQEPYRLKLSy I LERLHNT R DRNTRLQQQQEKDPTTPLPEYRDGTLYQAGTAF LE DLKLI QHNLKQTGLSCYELEKLI CQVE I FGFNLVHLDI RQESSRHS DAI NE i C ~47

# # # # # ## # ## #

Maize C~ M1 Tobacco M8 Fk~eda C4 Me k~pmt I M0 sombre c4 m

M6 C4~IK E RAQVTAN -YRELSEAE KLEVLLKE LRS PR PLI PHGS DEY S EVT DRE LG I FRTAS EAVKK FG PRMVPHC I I SMAS SVT DVLE PMVLLKE FGL IAAN GDN PRGTVDVI pL F m A J ~ EYLQI LpQpYNELSEAERTA~LVQELKTRRPLVP-ARMPFSE STREI I ETLRMVKQLQEE FGEAACQTYI i SMSRELSDLLEVLLLAKEVGLY DpVTG--KSSLQVI PLF

# # # ## ### # ### # # # # # ###

C ~ I T L ET I E DLQAC~GI L DE LWKI DLYRNYL- LQRDNVQEV~LGYS DSNKDGGY VSAN~ALYDAELQLVELCRSAGVKERL FHGRGGTVGRGGGPSYDAI LAQPRC~VQGSVRI T ~44 A31idu~ll~ ETVE DLQNAPRV~T~ FELP FYTQLN ?TQSEPLQEV~LGYS DSNKDSGFLSSN~E I HKAQKALGTVARDHRV~LR i FHGRGGSVGRGGGPAYE/~ LAQPGRTT DGR i KI T ~ 4

# ## # # # ## ##### ## # # # #####~ #### # # # ## # #

Maize CS ?74 Tobacco ;72 Flavefla C4 772 k=e p~.* 1 77~ Sorghum C4 E.coil 718 C . ~ u t a m . EQGZ I I SAKYGN PETARRNL~LVSATLEASLLDVS~LT DHQR~Y DI MSE I SE LS LKK~AS LVHE DQG F1Dy FTQSTPLQE i GSLN i GSRPSSRKQTS- SVE DLRAI PWV 7E3 AJIkiiuLlan8 EQGEVLASKYAL PELALYNLET I TTAVI QSSLLG- SGFDDI E PWNQIMEELAARS RRHY RALVYEQPDLVDFFNQVT FI EE I SKLQI SSRPARRKTGKRDLGSLKAI pWV

#~ ~ # # # ## # ### ~ ######

Figure 2.

930

Maize C3 Tobacco Flaveda C4 Ice pla.t t Sorghum C4 E.coll C.glutam. A.nidulans

LSWSQSRV/~ILPGWFGVGTALEQWI GEGEQATQRIAE LQT LNESWP FFTSVL DNI~Q'~IS K3LELRL3kKLYADLI PDTEVAER- - - \rY SVI REEY FLTKKM FCVI TGSDDLL S ~ FSW~QSRFLLPSWYGVGTALQE FLQ- - E RPEQNLNLLRY FYEWclP FFRI~v'I S ~ E MTLAKVDLQIAH HYVH£LANPE DQER FERVFSQIA,M~ FQLTCHLVLT I TNHGRLL g~ll

# # # ## # # # # ### # #

S p r u c e ~PTLK~aLRLRDSY Z ~ ~ I ~ I ~ ] ~ m U ~ H L S I ~ - - - SSTI~ AAELVKI~PT RyAFGLI~TLI L T I e ~

M~eC3 EGDLYLKQRLRLRDAY I TTLNVCQAYTLKRI RDPDY HVALRPHLSKE I M- DSTKAAADVVKLN[~GSEYAPGLE DTLI LTIIC~IAA~G

T o b a c c o EG DFY LKQRLRLRDSY I TT LN LLQAYTLKRI RDPNY HVT LRPH I SKDYM-ES- KS AAELVQLN PTSEYAPGLE DTL I LT~m~ZA~

F ~ C 4 EGDPY LKQGI RLRDPY I TTLNVCQAYTLKR I RDPNYHVT LRPH I SKEYAAE PSKF ADELI H LN PTS EYAPGLE DTLI LTlfd3I~

~ I EGDPYLKQRLRLRDPY!TTLNVCQAYTLKRIRDPDFKVTERPHLSKEIM-DAHKAAAELVKLNPTSEYAPGLEDTLI L T M ~

~ EGDPY LKQGLRLRN PY I T TLNVFQAYTLKR I RDPS FKVT PQPPLSKE EADENWPA GLVKLN - - -GERVPPGLE DT L I L ~ ~ M

F.COJ| ADL PWIAE S I QLRN I YT DPLNVLQAELLH RSRQAE ........................... KEGQEPDPRVEQALM ~ X ~ n3

C.~]~ DDN PLLARSVQRRY PYLLPLNVI QVE~fi~RRYRKGDQ ................................ SEQVSRNIQ LTI~4GLIITAL~ ~1~

A . R ~ DGDPELQRSVQLRNGT IVPLG FLQVALLKRLRQYRQ ................... QTETTGLMRSRYSKGELLRGAL L T I N G ~ I~ # # # ~ # # # # #

Figure 2. Comparison of the deduced spruce PEPC polypeptide with various PEPCs from higher plants (maize [26], tobacco [28], Flaveria [21], ice-plant [14], Sorghum [36]) and procaryotes (E. coli [19l, C. glutamicum [16], A. nidulans [25]). The spruce PEPC (this work) and the conserved carboxy terminus of PEPC described by [hui] are emphasized in bold print. Conserved sites are marked by #. Gaps which were introduced for optimal alignment are represented by hyphens and gaps which are unambiguously shared between higher plant PEPCs and the E. coli enzyme are boxed.

coIi [16, 19, 25, 39, 46]. It seems to be an essential component serving the stability of the enzyme [23].

Southern analysis of genomic DNA

To estimate the number of spruce PEPC genes, we hybridized an c~32p-labelled fragment of the PAP- PC14/2 cDNA containing lkb of the coding region to genomic spruce DNA digested with AsnI, DraI and SspI (Fig. 3). Since none of the enzymes used cuts in the probe sequence, the number of detectable bands was minimized. After moderately stringent washing (1 × SSC, 0.1% SDS; 60 °C) all restriction digests revealed multiple fragments of different intensities when hybridizing to the probe. After high-stringency washing (0.1 × SSC, 0.1% SDS; 65 °C) the number of fragments detected was reduced. This result excludes the possibility of a partial digest. Under high stringency conditions only two bands were observed in AsnI digests. Digestion with DraI resulted in two major bands and one faint band under high stringency conditions. One majore band of about 3 kb and one faint band of about 3.4 kt remained in the SspI digest after high stringency washing. These results supports the conclu- sion that there are several PEPC related sequences in the genome of spruce.

Analysis of PEPC mRNA by northem blot hybridization

To study the expression of the spruce PEPC gene in roots of seedlings and in cotyledon whirls, steady-

state levels of PEPC mRNA were measured by northern blotting (Fig. 4). Since in previous experiments with poly(A) + RNA isolated from adult needles no ppc specific signal had been obtained by northern blotting (our unpublished results), we incorporated RT-PCR products of the rbcS gene family as control probes in northern blotting experiments. The choice of this control was particularly good, because the rbcS mRNA is a lot shorter and thus the signals in the autoradiogram did not overlap. The size of the PEPC mRNA is approximately that of the 25S rRNA, independent of the tissue from which it was extracted. While the cotyledon poly(A) + RNA produced only a very faint band corresponding to PEPC mRNA, the transcript was abundant in roots. The rbcS mRNA, on the other hand, was strongly expressed in cotyledons, whereas only a faint signal was obtained with root poly(A) + RNA. In combination, these results indicate that the poly(A) + RNA was intact in both lanes. After stringent washing of the blot both signals, the ppc-specific as well as the rbcS-specific signal, became weaker.

Phylogenetic analysis

We analyzed the phylogeny of PEPCs using the neighbour-joining (NJ) method (Fig. 5). The analysis shows that the spruce PEPC is clustered with all monocot and dicot C3-form PEPCs used in the analysis with one exception: the Sorghum CP21 sequence. The multifurcation, which is not resolved by our bootstrap analysis, includes the C4-form PEPC of the dicot Flaveria trinervia, which is grouped together with the

931

Table 1. Conservation of PEPC protein sequences (% identities). The deduced amino acid sequences were aligned and compared individually using the CLUSTAL W Program [59]. All sites with gaps(in any sequence) were deleted and the remaining 829 amino acids were used for comparison.

Spruce Tobacco Sorghum Maize Flaveria Ice-plant Sorghum Maize E. coli b C. glutamicum b

C3 a C3 b C3b c C3 b C4 b CAM b C4 b C4 d

A. nidulans b 36 36 36 36 35 36 37 36 35 34

C. glutamicum 32 33 33 32 33 31 33 33 34

E. coli 44 43 44 44 43 43 43 43

Maize C4 79 80 80 80 79 78 92

Sorghum C4 76 77 78 77 76 75

Ice-Plant CAM 82 83 85 85 81

Flaveria C4 84 86 86 86

Maize C3 87 88 98

Sorghum C3b 88 88

Tobacco C3 86

aSpruce sequence reported in this paper, bFor references for tobacco C3-form, maize C3-form, E trinervia C4-form, ice-plant inducible CAM- form, Sorghum C4-form. E. coli, C. gutamicum and A. nidulans sequences, see Fig. 2. CSorghum C3-isoform CP 28 [35] aMaize C4-form PEPC [231.

Figure 3. Southern blot analysis of spruce genomic DNA digested with AsnI (lane 1), DraI (lane 2) and SspI (lane 3). The digests were electrophoresed through a 0.8% agarose/0.5x TBE gel and blotted onto positively charged nylon membranes. The blots were hybridized with the 32p-labelled 1.0 kb SspI fragment of PAPPC 14/2 cDNA and washed under various conditions. A. Blots washed under low-stringency conditions (60 °C, I x SSC). B. Blots washed under high stringency conditions (65 o C, 0.1 x SSC). The sizes indicated (in kb) were derived from molecular weight markers. Arrows indicate the positions of hybridizing bands.

932

Figure 4. Autoradiogram showing hybridization of spruce mRNA with the 32p-labelled full-length PEPC cDNA of spruce. Poly(A) + RNA (1 #g per lane) from roots (lane R) and cotyledons (lane C) was separated in a 1.0% agarose gel under denaturing conditions and blotted onto positively charged nylon membrane. 32p-labelled PCR products of spruce rbcS-specific cDNAs were also added to the hybridzation solution to serve as an internal control for the blotting procedure and for intactness of the RNA. The mobilities of the 25S and 18S nuclear-encoded RNA are shown.

C3-form PEPCs of Flaveria pringlei, the ice-plant and the two Solanaceae sequences of tobacco and potato. When we exclude the PEPC of F. pringlei from the analysis, the C4-form PEPC of F. trinervia is shifted towards the monocot C4-form PEPC (data not shown).

The plant PEPCs are more related to the E. coli PEPC (which is a member of the "7 subdivision of purple bacteria sensu Woese [62]) than they are to the PEPC sequences of gram positive Coryne- bacterium glutamicum and the two cyanobacterial sequences of Anabaena variabilis and Anacystis nidulans. Except for the above mentioned shift of the PEPC of E trinervia and the addition of spruce PEPC, our phylogenetic tree is almost identical to that obtained by Toh and coworkers [58], who also used the neighbour- joining method in their analysis.

Discussion

Phosphoenolpyruvate carboxylase has been studied at the molecular level most extensively in angiosperms, but little is known with respect to gymnosperms. This report presents a full-length cDNA sequence coding for PEPC from a coniferous tree species. This cDNA clone will be valuable for the understanding of the molecular mechanisms underlying gene regulation in gymnosperms as well as the evolutionary relationships

between PEPCs of angiosperm and gymnosperm species.

The amino acid sequence deduced from the clone PAPPC14/2 shows high sequence similarity to the angiosperm counterparts with the exception of monocot C4 forms (Table 1). The results thus confirm earlier indications of the conserved nature of PEPC. C3- form PEPC appears to be quite resistant to evolutionary change. Amino acid alignments reveal a high degree of similarity and colinearity [21, 36, 58]. This view is in particular supported by the conservation of the phosphorylation motif Arg/Lys-X-X-Ser in the PEPC of spruce (Fig. 1). This motif is present in all plant PEPC sequences analysed so far, suggesting the presence of this regulatory domain in the common ancestor of angiosperms and the gymnosperm family of Pin- aceae. Since findings in vitro as well as in vivo confirm that not only the PEPCs of C4 plants but also those of C3 plants [15, 53] can be phosphorylated, it can be assumed that the activity of spruce PEPC is modulated the same way.

The 5' leader sequence of the cDNA (see Fig. 1) contains a small upstream open reading frame (sURF). Such sURFs can be found in 5-10% of all eukaryotic mRNAs [29] and are assumed to influence the translation efficiency of the ORFs located downstream. Often they inhibit downstream translation in proportion to the efficiency of their own translation, which is, among other factors, dependent on the sequence context of their start codon [31]. Since the sequence context of the start codon of the sURF of the ppc mRNA dif- fers only in two bases from the consensus sequence for plant mRNAs [38], this sURF can be expected to contribute to the regulation of spruce PEPC synthesis at the level of translation.

There are two ATG triplets in the PEPC encoding ORF that are candidates for translation initiation (see Fig. 1). Two possible start codons are also found in the ppc2 gene of the ice plant [13] (as corrected by Albert and coworkers [1]), as well as in the PEPC cDNAs of soybean [56] and alfalfa [48]. In any of these PEPCs, when translation is alternatively initiated from the second ATG triplet the basic amino acid in their phosphorylation motif Lys-Met-Ala-Ser would not be present to guide the recognition by protein kinase [56]. Although it appears likely that translation is initiated from the first ATG triplet, as has been ascertained for eukaryotic translation in general [30], the use of both codons cannot be ruled out. The N-terminal sequencing of purified PEPC of the mentioned species could finally

933

100 1°° i loo E

:LO~ [ _ _

Tobacco C3 Ice-plant C3 F.trinervia C4 F.pringlei C3

IS~ruce C31 Sorghum C3b Maize C3 Sugarcane C3 Ice-plant CAM Sorghum C3a Sorghum C4 Maize C4

I E.coli I C.glutamicum I A.variabilis

loe I [A.nidulans

! !

0 0.1

Figure 5. Phylogenetic tree of phosphoenolpyruvate carboxylase (PEPC) amino acid sequences. The spruce PEPC and the prokaryotic PEPCs are boxed. Branch length and tree topology were estimated using the neighbour-joining method [50]. Values on the branches indicate percentage support obtained from 100 bootstrap replications for the cluster to the right of that value. Branch lengths are drawn to scale. The scale bar indicates 0.1 amino acid substitutions per site. The following sequences were added to the sequences described in Fig. 2 and Table 1 : ice-plant C3-form [13], E pringlei C3-form [21] and Sorghum C3-isoform CP21 [35].

settle the question which of the two codons is used to start the translation in vivo.

Our northern experiments show that the mRNA corresponding to the cDNA clone PAPPC 14/2 is present at a higher steady-state level in roots of seedlings than in the photosyntheticly active tissue of cotyledons (Fig. 4) and adult needles (data not shown). The difference in the contents of ppc-specific transcripts in these tissues may be explained by the physiological function of this enzyme in conifers. It is commonly accepted that conifers assimilate nitrogen in their roots [41] and that PEPC is the enzyme of carbon metabolism primar- ily regulated by nitrogen availability [55]. In addition, Wingler and coworkers [61] have demonstrated that PEPC activity in roots of spruce seedlings is higher than in the corresponding cotyledons under most of the nitrogen conditions they used in their experiments. Based on these data it can be suggested that PEPC is a basic component of the N-fixation cascade in the roots of spruce and that the high PEPC activity is attended by an enhanced expression of the ppc gene corresponding to the PAPPC 14/2 cDNA and/or a higher ppc mRNA stability in this tissue. However these findings do not imply that phosphorylation is not a means of regulat- ing the in vivo activity of spruce PEPC. In addition the lower response of the PEPC cDNA with cotyledon and needle mRNA could simply reflect the presence

of ppc-mRNAs with a lower level of homology to the probe cDNA.

In the C4 plants maize [22] and sorghum [11] as well as in the facultative CAM plant Mesembry- anthemum cristallinum [12] PEPC is encoded by a small gene family. The result of our genomic South- em blot analysis indicates that beside the gene coding for the PAPPC14/2 mRNA there are other ppc related sequences in the genome of the Norway spruce (Fig. 3). Although PEPC gene families have not been characterized in C3 plants as yet, previously published results suggest that varying PEPC isoforms may be present in C3 plants [43, 48, 54]. At present, we do not know if the additional sequences are function- al PEPC genes, PEPC pseudogenes and/or otherwise PEPC-related sequences. Because of the high level of genetic diversity of conifers, PEPC alleles may also contribute to the complex banding pattern observed in Southern analysis. Although the northern blot data did not reveal the existence of multiple ppc mRNAs, the low-level expression of additional ppc genes or the presence of inducible ppc genes induced by external stimuli cannot be ruled out.

Examining the relationship between spruce PEPC and previously published PEPCs of angiosperms and prokaryotes, we performed a phylogenetic analysis employing the neighbour-joining method (see Fig. 5). In the resulting tree the spruce PEPC is grouped, in a

934

multifurcation, with all the C3-form PEPCs of monocot and dicot species used in the analysis, the only exception being the sorghum CP21 PEPC. These results are in contrast to previously published results based on rRNA [60] and protein [6, 40, 47, 64] sequence data and revealed that the sequences of Pinaceae form a group well-removed from the dicot and monocot sequences. However, the differences in the gene or amino acid sequences between these three groups of seed plants are rather small. Martin and coworkers [42] have estimated based on of gapC and rbcL sequence data that the monocot-dicot split occurred about 300 million years ago, only about 30 million years after the conifer-angiosperm divergence (about 330 million years ago). Although their time scale has been cri- ticized by many authors, the monocot/dicot split a short time after the conifer-angiosperm divergence is increasingly supported by various sequence analyses. Since both points of divergence almost coincide in evolutionary time, the phylogenetic analyses of slowly evolving proteins, as in the case of the housekeeping forms of PEPC, may not always unveil the true relationships between the different groups of Spermatophyta. However, the trichotome branching order is another hint pointing to a close phylogenetic relationship of seed plants.

The evolutionary origin of plant PEPC is still a mat- ter of debate. One view is that plant PEPC may have arisen from an endosymbiotic ancestor of chloroplasts [37]. This hypothesis would satisfactorily explain why this enzyme is only found in the eukaryotic line lead- ing to higher plants. However, the fact that PEPCs of cyanobacteria and plants are distantly related to each other could be evidence against the theory of a plastid origin of plant PEPCs. Still another scenario results when we consider the relatively close relationship between the PEPC of E. coli and the plant PEPCs, already ascertained in earlier analyses [23, 36, 37, 58]. The E. coli PEPC shares a higher degree of identity with plant PEPCs (43-44%) than with other published prokaryotic sequences (31-37%). This close relationship is accentuated not only by the degree of homology (Table 1 ) but also by the position in the gene tree (Fig. 5) and in the alignment of the PEPCs (Fig. 2). In comparison to the other prokaryotic sequences, the sequences of E. coli and plants can be considered almost colinear. E. coli is a member of the "7 subdivision of purple bacteria sensu Woese [62]. Even today many purple bacteria species live in symbiosis with eukaryotic organisms. The data actually suggest a scenario in which the ppc genes of plants diverged

from a common ppc gene transferred to the nucleus from ancestors of present-day purple bacteria. Since the purple bacteria are regarded as the precursors of mitochondria [63], the ppc gene may very well also derive from this organelle. In any case, the divergence point ofE. coli and plant PEPCs is rooted deep enough in the tree to support this view. Yet, further prokaryotic sequences are necessary to verify the above statements about the evolutionary origin of plant PEPC.

Acknowledgements

This work was supported by the Deutsche Forschungs- gemeinschaft (DFG) and by a doctoral grant from Rheinland-Pfalz. The authors are thankful to Profess- or E.R. Schmidt and Dr. Thomas Hanketn (Institut ftir Molekulargenetik der Universit~it Mainz, Germany) for her guidance in molecular evolution and for com- puter aided-search of spruce pappc 14/2 homologues in EMBL databases. We also thank Dr G. Igloi (Insti- tut fiir Biologie III der Universit~it Freiburg, Germany) for providing us with the ppc primers. We are also indebted to Dr H. Dilly-Hartwig and K. Kehl for help- ful discussions.

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