Molecular Phylogenetics and Evolution Vol. 20, No. 2, August, pp. 262–274, 2001 doi:10.1006/mpev.2001.0973, available online at http://www.idealibrary.com on New Partial Sequences of Phosphoenolpyruvate Carboxylase as Molecular Phylogenetic Markers Hans Gehrig,*,1 Valentina Heute,† and Manfred Kluge† *Smithsonian Tropical Research Institute, Unit 0948, APO AA 34002-0948, Panama; and †Darmstadt University of Technology, Schnittspahnstr. 10, 64287 Darmstadt, Germany Received October 10, 2000; revised March 12, 2001 and Chase, 1995; Yukawa et al., 1996; Qiu and Palmer, To better understand the evolution of the enzyme 1999). Because the obvious limitation in the assort- phosphoenolpyruvate carboxylase (PEPC) and to test ment of suitable markers may be one reason for con- its versatility as a molecular character in phylogenetic troversial interpretations of obtained results, it is and taxonomic studies, we have characterized and highly desired that more markers become available for compared 70 new partial PEPC nucleotide and amino taxonomic and phylogenetic studies in plant sciences. ؅ acid sequences (about 1100 bp of the 3 side of the Searching for such markers, we investigated and com- gene) from 50 plant species (24 species of Bryophyta, 1 pared sequences of phosphoenolpyruvate carboxylase of Pteridophyta, and 25 of Spermatophyta). Together (PEPC; EC 4.1.1.31). with previously published data, the new set of se- PEPC catalyzes the ␤-carboxylation of phosphoenol- quences allowed us to construct the up to now most pyruvate, with oxaloacetate and inorganic phosphate complete phylogenetic tree of PEPC, where the PEPC sequences cluster according to both the taxonomic as products (Utter and Kolenbrander, 1972; Andreo et positions of the donor plants and the assumed specific al., 1987). The enzyme is ubiquitous in prokaryotic function of the PEPC isoforms. Altogether, the study microorganisms and plants, and it is involved in many further strengthens the view that PEPC sequences can functions including photosynthetic and anaplerotic provide interesting information for the reconstruc- CO2 fixation (e.g., Kluge and Ting, 1978; Winter, 1985; tion of phylogenetic relations between organisms and Cushman and Bohnert, 1999; Latzko and Kelly, 1983), metabolic pathways. To avoid confusion in future dis- production of carbon skeletons in symbiotic nitrogen cussion, we propose a new nomenclature for the deno- fixation (Schuller et al., 1990), modulation of turgor in tation of PEPC isoforms. © 2001 Academic Press stomatal guard cells, maintenance of ion balance, pH Key Words: crassulacean acid metabolism (CAM); state mechanisms, and others (Latzko and Kelly, 1983; photosynthesis (C3,C4); molecular taxonomy; molecu- Melzer and O’Leary, 1987). In most bacteria and plants lar evolution; phosphoenolpyruvate carboxylase studied so far, physiological and molecular approaches (PEPC). showed the existence of PEPC multigene families (Cushman and Bohnert, 1989a,b; Cre´tin et al., 1991; Poetsch et al., 1991; Kawamura et al., 1992; Lepiniec et INTRODUCTION al., 1993, 1992; Gehrig et al., 1995) encoding function- and tissue-specific isoforms of the enzyme (Lepiniec et The comparison of organisms on the level of molec- al., 1994; Toh et al., 1994; Rajagopalan et al., 1994; ular characters has become a powerful and now indis- Gehrig et al., 1998b). Because of the ubiquitous distri- pensable tool in taxonomic and phylogenetic research. bution of PEPC and the high diversity in its functions However, the unequivocalness of results obtained by it has been proposed that the nucleotide sequences of this approach depends essentially on the availability of the PEPC genes and the amino acid sequences of the versatile molecular markers. In plant sciences mainly gene products should provide powerful markers in four types of nucleotide sequences are used as such molecular taxonomic and phylogenetic investigations markers, namely the 18s rRNA (e.g., Bopp and Cape- (Gehrig et al., 1998a). sius, 1996; Qiu and Palmer, 1999), ITS regions (e.g., First attempts to construct phylogenetic trees of Bruns et al., 1991; Bogler and Simpson, 1996), MADS- PEPC were based on full-length sequences (Lepiniec et box genes (e.g., Winter et al., 1999), and the rbcL genes al., 1993; Toh et al., 1994; Cushman and Bohnert, coding for the large subunit of RUBISCO (e.g., Dressler 1996; Honda et al., 1996). Gehrig et al. (1998a) showed 1 To whom correspondence and reprint requests should be ad- that the comparison of partial PEPC sequences can dressed. Fax: (00507)-212-8148. E-mail: [email protected]. provide valuable information on the phylogenetic in- 262 1055-7903/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. PHOSPHOENOLPYRUVATE CARBOXYLASE AS MOLECULAR MARKER 263 terrelationships of PEPC isoforms and the donor tained was modified by visual inspection to increase plants from which the genes were isolated. Although the total alignment score (the alignment is available up to now full-length and partial PEPC nucleotide from the authors). Sequence data were evaluated sequences of 11 prokaryotes (11 PEPC sequences), 1 by means of the PHYLIP package, version 3.5c (Fel- alga (2 PEPC sequences), 5 ferns (5 PEPC sequences) senstein, 1993). Neighbor-joining analysis (Saitou and 25 higher plants (48 PEPC sequences) have been and Nei, 1987) was employed as a distance method determined (Toh et al., 1994; Chollet et al., 1996; (PROTDIST) with 1000 resamplings with the Kimura Gehrig et al., 1998a) our present knowledge on the formula for amino acid sequences (Kimura, 1983) of the phylogeny of PEPC sequences and the isoforms in spe- PHYLIP package. This is a rough-and-ready distance cies is still quite fragmentary and urgently requires formula for approximating PAM distance that simply completion. For this reason we have analyzed PEPC measures the fraction of amino acids that differs be- sequences in numerous further plant species including tween two sequences. Parsimony analyses with 100 Bryophyta, Pteridophyta, and Spermatophyta. The resamplings were done with the PROTPARS program present paper reports on the obtained results. of the PHYLIP package. This program infers an un- rooted phylogeny directly from protein sequences (for MATERIALS AND METHODS further explanations see Felsenstein, 1993). In all cases Escherichia coli was used as outgroup. Plant Material The experimental plants were cultivated in the Bo- RESULTS AND DISCUSSION tanical Gardens of Heidelberg and Darmstadt (Ger- many). The plant material was thoroughly cleaned As already mentioned in the Introduction, the PEPC with sterilized water and by ultrasonic treatment. Af- is coded by multigene families, with isoforms being ter the cleaning, tissue samples for RNA preparation linked to a wide range of different functions. Up to now were immediately frozen in liquid nitrogen and stored there has been no generally followed terminology of at Ϫ80°C until further processing. PEPC sequences suitable for relating a given PEPC isoform to a specific function. However, to avoid confu- RNA Extraction and PCR Amplification sion, definition of a common nomenclature to denote Total RNA was extracted with the guanidine isothio- PEPC isoforms is highly desired. Thus, we propose to cyanate method (Chirgwin et al., 1979) or with the distinguish and to denote PEPC isoforms as follows QIAGEN plant RNA isolation kit (Qiagen, Germany), (Table 2): prokaryotic anaplerotic and other nonphoto- depending on the plant material. RNA quality was synthetic isoforms (ppc-aP), eukaryotic anaplerotic and examined by agarose gel electrophoresis, and after re- nonphotosynthetic isoforms (ppc-aX; with X standing verse transcription an approx 1100-bp PEPC fragment for R ϭ root, aerial root, root nodule, and for L ϭ leaf), was amplified by RT-PCR. The RT-PCR was performed and photosynthetic isoforms catalyzing the primary with two degenerated PEPC primers [PEPC1: TC- CO2 fixation in C4 photosynthesis and crassulacean (CTA) GA(TC) TC(CAT) GG(AC) AA(AG) GA(TC) GC; acid metabolism (CAM) (respectively, ppc-C4 and ppc- PEPC2: GC(GAT) GC(GAT) AT(GCA) CC(CT) TTC CAM). The term “C3” isoform often used in the litera- AT(GT) G] under the following conditions: 35 cycles ture to describe anaplerotic or “housekeeping” PEPC (Personal Cycler; Biometra, Germany) at 95°C for 30 s, isoforms in leaves of C3 plants (e.g., Lepiniec et al., 55°C for 30 s, 72°C for 3 min. The PCR products were 1994; Toh et al., 1994; Rajagopalan et al., 1994) should cloned into the TA vector system of Invitrogen (Neth- be avoided, because PEPC is not directly involved in erlands). Different PEPC isoform clones of the Kalan- the C3 pathway of photosynthesis. In the present paper choe and orchid species were identified by digestion we will follow the terminology proposed here. with BamHI, HindIII, PstI, SalI, and EcoRI and ana- In the context of the question whether PEPC can lyzed on 0.8% agarose gels. serve as a useful molecular marker in taxonomic and Randomly selected transformants of each amplified phylogenetic investigations, in the present study we PEPC cDNA clone were sequenced in both directions have analyzed numerous new partial PEPC sequences. (SeqLab Co., Hannover, Germany). The nucleotide se- The results not only increase the number of plants quence data have been submitted to the EMBL and species that can be compared on the level of PEPC GenBank nucleotide sequence databases (see Table 1 sequences but also include more taxa that are sup- for accession numbers). posed to mark branching points of plant evolution. Altogether, in the present study 70 new PEPC se- Sequence Analysis quences were analyzed and documented in the EMBL Amino acid sequences were obtained from 143 nucle- gene bank (Table 1). The new sequences represent 24 otide PEPC sequences. The alignment and sequence species of Bryophyta, 1 species of Pteridophyta, and 25 identity were calculated for each pair with the Align- species of Spermatophyta. ment Editor 3.7 (Hepperle, 1997). The alignment ob- As previously done (Gehrig et al., 1998a), in the 264 GEHRIG, HEUTE, AND KLUGE TABLE 1 PEPC Partial Sequences Included in the Calculations of the Phylogenetic Trees Shown in Figs.