Isolation of a novel class of bZIP transcription factors that interact with ABA-responsive and embryo-specification elements in the Dc3 promoter using a modified yeast one-hybrid system

Dc3 is a carrot lea class gene that is abundantly expressed during somatic and zygotic embryogenesis. Its expression is normally embryo-specific and also can be induced by abscisic acid. The regulatory elements mediating the embryo-specific expression of Dc3 reside within the proximal promoter region (-117 to +26), which is also essential for ABA-induced expression. In this study, an optimized version of the yeast one-hybrid system has been used to clone factors that bind to the promoter region of the Dc3 gene. Twenty-five million yeast transformants were screened in a single experiment, and nine independent cDNA clones were isolated from a sunflower library that encode proteins that specifically bind to functional cis-regulatory elements in the Dc3 promoter. Analysis of these clones showed that they are derived from three different mRNA species that encode two basic leucine zipper proteins. The basic regions of these proteins, named DPBF-1 and 2 (Dc3 Promoter-Binding Factor-1 and 2), respectively, are nearly identical to each other and are similar to the plant G-box binding factor GBF-4. Outside the basic region, however, DPBF-1 and 2 diverge significantly from each other and from other known factors. Both factors have transcriptional activity in yeast, and bind to DNA as dimers. Unlike other plant bZIP factors, DPBF-1 and 2 recognize sequences containing the ACACNNG core. Cloning of these factors demonstrates the power of the one-hybrid approach when optimally applied.