Enhancer-mediated reporter gene expression in Arabidopsis thaliana: a forward genetic screen.

Gene expression is controlled and regulated by interactions between cis-regulatory DNA elements (CREs) and regulatory proteins. Enhancers are one of the most important classes of CREs in eukaryotes. Eukaryotic genes, especially those related to development or responses to environmental cues, are often regulated by multiple enhancers in different tissues and/or at different developmental stages. Remarkably, little is known about the molecular mechanisms by which enhancers regulate gene expression in plants. We identified a distal enhancer, CREβ, which regulates the expression of AtDGK7, which encodes a diacylglycerol kinase in Arabidopsis. We developed a transgenic line containing the luciferase reporter gene (LUC) driven by CREβ fused with a minimal cauliflower mosaic virus (CaMV) 35S promoter. The CREβ enhancer was shown to play a role in the response to osmotic pressure of the LUC reporter gene. A forward genetic screen pipeline based on the transgenic line was established to generate mutations associated with altered expression of the LUC reporter gene. We identified a suite of mutants with variable LUC expression levels as well as different segregation patterns of the mutations in populations. We demonstrate that this pipeline will allow us to identify trans-regulatory factors associated with CREβ function as well as those acting in the regulation of the endogenous AtDGK7 gene.