Salt stress alters A/T-rich DNA-binding factor interactions with the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum
1992
Cushman, J.C. | Bohnert, H.J.
The common ice plant, Mesembryanthemum crystallinum, shifts from C3 to crassulacean acid metabolism (CAM) photosynthesis in response to osmotic stress. The expression of a number of genes encoding enzymes involved in the CAM pathway increases as a result of increased transcription rates. To begin to investigate the mechanisms responsible for the transcriptional activation, we have characterized the 5' control region of a specific isoform of phosphoenolpyruvate carboxylase gene (Ppc1) that plays a key role in CAM. We have determined the nucleotide sequence of the 5' flanking region of this gene. Ppc1 contains a long 5'-leader sequence with the transcriptional start site located 332/333 nucleotides 5' of the translational initiation codon. Multiple DNA interactions with nuclear factors are detectable within the 5'-flanking region of Ppc1. We have used copper orthophenanthroline footprinting to demonstrate that one particularly abundant factor (designated PCAT-1) binds the Ppc1 promoter at two distinct A/T-rich sites located -128 to -158 and -187 to -205 bp upstream of the transcriptional start site. These binding sites share a loose consensus motif having the sequence AARTAAC(T/A)A(G/T)TTTY. Gel retardation competition experiments with oligonucleotides containing these A/T-rich binding sites suggest that both sites bind the same factor, but with different affinities. Fractionation of crude nuclear extracts by heparin-agarose chromatography indicates that PCAT-1 is more prevalent in extracts prepared from salt-stressed leaf tissue. Additional binding activities that interact with the PCAT-1 binding sites have been detected that either increase or decrease in abundance or binding affinity in response to salt stress.
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