Promoter activation by ACGT in response to salicylic and abscisic acids is differentially regulated by the spacing between two copies of the motif

A variety of small sequence motifs, located upstream of minimal promoters influence gene expression. The function of the core sequence ACGT, present in a family of commonly occurring cis acting transcription regulatory motifs was investigated in the background of an artificially designed synthetic promoter sequence. The ACGT was placed in one or two copies and separated by different spacer lengths between the two copies, to study their affect on the expression of basal promoter in plant cells. The activation of transcription by the ACGT element was examined by transient and stable transformation in tobacco, using gusA as the reporter gene. The analysis shows that the expression of the reporter gene was influenced differently by spacing between two adjacent copies of the motif. Two copies of the ACGT element separated by 5 nucleotides gave highest activation. This configuration imparted salicylic acid inducibility to the basal promoter. However, two copies of ACGT separated by 25 nucleotides allowed the promoter to be induced by abscisic acid but not salicylic acid. Computational analysis of the Arabidopsis thaliana genome database showed the presence of these motifs in several genes associated with a variety of stress responses. The results on motif-related inducibility by salicylic acid and abscisic acid, as seen in a synthetic sequence background were validated by experiments on the expression of the native promoter of a protein phosphatase 2C-like gene of Arabidopsis in tobacco leaves. The study supports the importance of spacing between the ACGT sequence motifs on the elicitor specific modulation of gene expression and demonstrates the role of different ACGT motifs in the regulation of PP2C gene expression.