Two single-base substitutions involved in altering in a paired-box of AAATAC in the promoter region of soybean lipoxygenase L-3 gene impair the promoter function in tobacco cells

Abstract We have characterized the lox3 gene, a mutant form of Lox3 that encodes soybean seed lipoxygenase L-3. L-3 null mutants exhibit no detectable accumulation of the Lox3 transcript. Nucleotide sequencing of the 5′-flanking regions (−861 to +1 of the translation initiation site) of Lox3 and lox3 revealed that three substitutions (mut1, mut2 and mut3) are unique in the mutants. Interestingly, mut1 and mut2 are involved in changing a paired-box of AAATAC. The promoter region of either Lox3 or lox3 was fused to the coding region of the β-glucuronidase (GUS) gene and these fusion genes were expressed transiently in tobacco suspension-cultured cells. Gross reduction of GUS activity in the tobacco cells carrying the GUS gene driven by the lox3 promoter was observed. Repair of either mut1 or mut2 to wild type in the fusion gene by site-directed mutagenesis resulted in the recovery of GUS activity to the wild-type level, while repair of mut3 to wild type had less effect on the GUS gene expression. These results indicate that mut1 and mut2 occur in redundant cis -acting elements, probably in the paired-box, suggesting the molecular basis for the L-3 null mutation.