title: Identification of enhancer and silencer regions involved in salt-responsive expression of Crassulacean acid metabolism (CAM) genes in the facultative halophyte Mesembryanthemum crystallinum.
abstract: In response to salinity or drought stress, the facultative halophyte Mesembryanthemum crystallinum will switch from C3 photosynthesis to Crassulacean acid metabolism (CAM). During this switch, the transcription rates of many genes encoding glycolytic, gluconeoagenic, and malate metabolism enzymes are increased. In particular, transcription of the Ppc1 and Gap1 genes encoding a CAM-specific isozyme of phosphoenolpyruvate carboxylase and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively, is increased by salinity stress. To investigate the molecular basis of salt-induced gene regulation, we examined the Ppc1 and Gap1 promoters for cis-elements and trans-acting factors that may participate in their expression. Ppc1 or Gap1 promoter-beta-glucuronidase chimeric gene constructs containing various deletions were introduced into intact, detached M. crystallinum leaves by microprojectile bombardmen. The Ppc1 5'-flanking region contains several salt-responsive enhancer regions and one silencer region reflecting the complex regulation patterns exhibited by this promoter in vivo. A region localized between nucleotides -977 and -487 relative to the transcriptional start site appears to regulate the magnitude of salt-inducibility. In contrast, the Gap1 promoter contains a single region from -735 to -549 that confers salt-responsive gene expression. Alignment of these 5'-flanking regions reveals several common sequence motifs that resemble consensus binding sites for the Myb class of transcription factors. Electrophoretic gel mobility shift assays indicate that both the -877 to -679 region of Ppc1 and the -735 to -549 region of Gap1 form a DNA-protein complex unique to nuclear extracts from salt-stressed plants. The appearance of this DNA-protein complex upon salt stress suggests that it may participate in salt-induced transcriptional activation of Ppc1 and Gap1.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454, USA.
authors.forename: H J
authors.initials: HJ
authors.lastname: Schaeffer
authors.forename: N R
authors.initials: NR
authors.lastname: Forstheoefel
authors.forename: J C
authors.initials: JC
authors.lastname: Cushman
mesh_terms: D000222:Adaptation, Physiological / Q000235:genetics*; D001483:Base Sequence; D004252:DNA Mutational Analysis; D018274:Electroporation; D004742:Enhancer Elements, Genetic / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics; D010788:Photosynthesis / Q000235:genetics*; D010944:Plants / Q000187:drug effects / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D014170:Transformation, Genetic
chemical_list: D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D009243:NAD; D012965:Sodium Chloride; D010730:Phosphoenolpyruvate Carboxylase