Regulatory regions and nuclear factors involved in nodule-enhanced expression of a soybean phosphoenolpyruvate carboxylase gene: implications for molecular evolution.

We have determined the genomic organization of two closely related phosphoenolpyruvate carboxylase genes in soybean, GmPEPC7, which is expressed at high levels in root nodules, and the housekeeping gene GmPEPC15. Their nucleotide sequences, including most introns and 5;-flanking regions within 600 bp upstream from the transcription start sites, are well conserved, suggesting that they were duplicated quite recently. To gain insights into the process of evolution of the tissue-specifically expressed GmPEPC7gene, we produced chimeric constructs carrying either the GmPEPC7or GmPEPC15promoter fused to the beta-glucuronidase gene. The expression patterns of the reporter observed in nodules that developed on transgenic hairy roots reflected the levels of mRNA levels produced by the genes in wild-type soybean plants, indicating that the GmPEPC7promoter directs nodule-specific expression. Loss-of-function experiments showed that the segment of GmPEPC7between -466 and -400, designated as the "switch region" (SR), was necessary for expression in nodules, although proteins that bind to SR were not detectable in a gel-retardation assay. Another gel-retardation assay indicated that putative nodule nuclear proteins bind specifically to the region of GmPEPC7between -400 and -318, designated as the "amplifier region" (AR). Both SR and AR have characteristic sequences that are not found in the GmPEPC15promoter. Furthermore, experiments using hybrid promoters derived from GmPEPC15demonstrated that AR confers high-level expression in nodules only in combination with SR. When wild-type soybean plants were subjected to prolonged darkness and subsequently illuminated, the level of GmPEPC7mRNA in nodules decreased and then recovered. This study suggests that the acquisition of two interdependent cis-acting elements resulted in molecular evolution of the nodule-enhanced GmPEPC7gene.