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.

Molecular Genetics of Crassulacean Acid Metabolism

“…The region between -487 and -320 appears to suppress salt-inducibility, suggesting that a silencer element is present within this region. In contrast, the GapCl promoter is less complex, with the elements responsible for salt-inducible expression contained within a 186-bp region located between -735 and -549 (Schaeffer et al, 1995). The elements located between -675 and -549 seem to be responsible for salt-inducible gene expression, whereas sequences from -735 to -675 appear to control the amplitude of expression in both unstressed and stressed plants.…”

“…The coordinate expression of these genes in this species suggests they may share common cis-acting regulatory elements to mediate stress-inducible expression patterns. To investigate this hypothesis, Schaeffer et al (1995) developed a transient expression system based on microprojectile bombardment of detached M. crystallinum leaves that faithfully reproduces CAM-specific patterns of gene expression. The stress-inducible Ppcl promoter contains a complex set of elements, including severa1 redundant activator domains and one silencer region.…”

“…Regions of the Ppcl and GapCl promoters sufficient to confer salt inducibility form slow-mobility DNA-protein complexes that are more abundant following CAM induction (Schaeffer et al, 1995) may be the result of de novo protein synthesis following salt stress. Alternatively, the factor(s) within this complex have undergone a modification to cause greater DNA-binding affinity under stress conditions.…”

Growth and development of Mesembryanthemum crystallinum (Aizoaceae)

“…Our preliminary experiments indicate a complex interaction of cis-acting DNA sequences of promoters with trans-acting transcription factors (Cushman & Bohnert, 1992). It appears that sequences close to the start of transcription of the Ppc1 gene are essential for stress induction, but that overall promoter activity depends on sequences remote from the transcription start (Cushman & Bohnert, 1992 ;Scha$ ffer, Forsthoefel & Cushman, 1995). Among the regulatory elements which govern transcription and induction during stress in the Ppc1 promoter are elements that have, in other systems, been shown to respond to changes in plant-growth regulator concentrations (Cushman, Meiners & Bohnert, 1993 ;Thomas & Bohnert, 1993 ;Thomas, Smigocki & Bohnert, 1995).…”

Evidence that the induction of crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling

“…1996). The promoters of Ppcl and GdhI (the stress-inducihle CA^4-specific isogene of NAD-dependent glyceraldehyde 3-phosphate dehydrogenase) contain both salt and ABA-like tesponse eletnents (Cushtiian & Bohnert 1992;Schaeffer et at. 1994).…”