Expression of the CAM-form of phospho(enol)pyruvate carboxylase and nucleotide sequence of a full length cDNA from Mesembryanthemum crystallinum

Abstract

We have determined the complete nucleotide sequence of a full length cDNA encoding the Crassulacean acid metabolism (CAM) isogene of phospho(enol)pyruvate carboxylase (PEPCase). The cDNA clone, 3348 bp in length, was obtained from mRNA isolated from Mesembryanthemum crystallinum (common ice plant) which had undergone salt stress and subsequent induction of CAM. The long open reading frame encodes PEPCase (EC 4.1.1.31) with a predicted molecular mass of 110533 daltons. The deduced amino acid sequence of the ice plant PEPCase is most similar to that from maize having an amino acid identity of 74.9%. Sequence identity in corresponding regions of the PEPCase proteins from Escherichia coli and the cyanobacterium Anacystis nidulans are 41.4% and 33.5%, respectively. A compilation of the four amino acid sequences permitted the identification of phylogenetically conserved regions within the proteins which may play a role in the function of this important enzyme in plant metabolism. Gene specific probes from 3' coding and noncoding regions of the cDNA clone used to probe genomic Southern blots established that this PEPCase gene is present in one copy in the nuclear genome of M. crystallinum. Transcripts arising from this gene increase dramatically when M. crystallinum is irrigated with 0.5 M NaCl, a stress which induces this plant to switch the primary fixation of CO2 from C3 (Calvin cycle) to CAM mode. The salt-induced mRNA encodes a PEPCase isoform which is undetectable in plants in the C3 mode as demonstrated by Northern hybridization.