Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression.
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.
Kim
D J
DJ
Institute of Cell and Molecular Biology, University of Edinburgh, Scotland.
Smith
S M
SM
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018553:Cucumis sativus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D017343:Genes, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D018749:RNA, Plant / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D012333:RNA, Messenger; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; D010729:Phosphoenolpyruvate Carboxykinase (GTP)