Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression

Abstract

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.

Sugar-Starvation-Induced Changes of Carbon Metabolism in Excised Maize Root Tips

“…In germinating seedlings and in senescing leaves, the glyoxylate cycle is associated with gluconeogenesis (Gut and Matile, 1988). The observation that glyoxylate cycle enzymes are induced, whereas PEP carboxykinase is not (Kim and Smith, 1994), suggested that in plant materials in which carbon export does not occur, the glyoxylate cycle may be used for biosyntheses other than gluconeogenesis. In a number of senescing or sugar-starved plant organs or cells, activities or mRNA amounts of malate synthase and isocitrate lyase, the two typical glyoxylate cycle enzymes, have been found to be increased (Graham et al, 1994, and refs.…”

“…it converts acetyl units to the four carbon organic acid succinate, thus allowing net biosyntheses from intermediates of the TCAC. In some cases, such as in senescing leaves, it appears to be linked with gluconeogenesis (Gut and Matile, 1988), but in detached leaves or in protoplasts, its function may be to provide four carbon precursors for amino acid synthesis (Graham et al, 1994;Kim and Smith 1994). In maize root tips only malate synthase was detected after starvation, and it was suggested that the role of this enzyme might be limited to the salvage of glyoxylate resulting from Gly catabolism (Dieuaide et al, 1992).…”

Purification, and phosphorylation in vivo and in vitro, of phosphoenolpyruvate carboxykinase from cucumber cotyledons

“…It also shows that the part of the enzyme which is cleaved by proteolysis (which may be the N-terminal extension) either contains the phosphorylation site or is necessary for efficient phosphorylation of the remainder of the molecule. Within the N-terminal extension, Ser 67 and Thr 68 are likely target residues for phosphorylation because, apart from being within a consensus site for recognition by mammalian cAMPdependent protein kinase [19], they are also within a sequence of amino acids, Gln-Lys-Lys-Arg-Ser-Thr (residues 63-68) [4], which is similar to a proposed phosphorylation site motif for plant PEP carboxylase-kinase (Glu/Asp-Lys/Arg-X-X-Ser, for review see [12]). The phosphorylation of PEPCK was reversible.…”

“…In cucumber, the gene for PEPCK codes for a protein with a predicted molecular mass of about 74 kDa [4]. All previous purifications of the enzyme from C3 and C4 plants have yielded an enzyme with a molecular mass of between 62 and 64 kDa [5,6].…”

“…These lower molecular mass forms arise in crude extracts by rapid proteolytic cleavage of forms with a higher molecular mass, of about 74 kDa in cucumber cotyledons and 68 kDa in the C4 grass, Urochloa panicoides [5]. The predicted amino acid sequence of PEPCK from cucumber is homologous to those from yeast, Rhizobium, Escherichia coli and trypanosomes [4], except that the enzyme from cucumber is considerably larger because it possesses an N-terminal extension of about 12 kDa [4], similar in size to the portion of the polypeptide which is lost after proteolysis. Several plant enzymes are known to contain N-terminal extensions when compared to their bacterial or Corresponding author.…”