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

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 109551. 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.

New insights into allelic diversity of a phosphoenolpyruvate carboxylase in the conifer Picea abies (L.) Karst.

“…Isolation of genomic PEPC sequences was done using long-range PCR with primer pairs that were sequence-deduced from the published cDNA of Norway spruce (Relle and Wild 1996;GenBank Acc. No.…”

“…This evidence arose from population genetic studies (Bergmann and Scholz 1989) as well as from in vitro studies of enzyme kinetics in Norway spruce (Rothe and Bergmann 1995). Facilitated by a published Norway spruce PEPC cDNA sequence (Relle and Wild 1996;GenBank Acc. No.…”

“…A subset of megagametophytes of the same seed mother was analysed for Mendelian segregation at a polymorphic restriction site. As the existence of a small PEPC DNA gene family cannot be excluded (Relle and Wild 1996), exon and intron sequence information was used to design de®ned probes for Southern analysis. The results were expected to shed light mainly on nucleotide variation among alleles of a gymnosperm gene and are discussed with regard to allozyme variation.…”

Photosynthetic and Other Phosphoenolpyruvate Carboxylase Isoforms in the Single-Cell, Facultative C4System of Hydrilla verticillata

“…These could be elements governing functional differences among the H. verticillata isoforms. In addition, there were two initiation codons downstream of the leader sequence, which are seen in other PEPC sequences (Relle and Wild, 1996). If translation is initiated from the second Met, then the motif upstream of the Ser residue is absent and the interaction of this residue with PEPC-protein kinase and the subsequent phosphorylation would not occur.…”

“…It is possible that the C 4 form of PEPC diverged before the monocot/ eudicot split 200 million years ago (mya) but after the gymnosperm and angiosperm divergence 330 mya (Wolfe et al, 1989;Relle and Wild, 1996). The PEPC from Norway spruce, which is suggested to be part of the N-fixation system in spruce roots (Relle and Wild, 1996) and, thus, is likely related to the pea root nodule PEPC, was potentially a sister to the H. verticillata isoforms and was closer to them than to other monocot C 3 or CAM PEPCs. If so, the H. verticillata PEPCs may represent ancestral sets of genes that emerged before angiosperm divergence and may provide clues to C 4 evolution in monocots.…”