A number of genes regulating leaf growth have been identified, often through gain- and loss- of function studies of single genes. Overexpression studies usually employ strong constitutive promoters such as the cauliflower mosaic virus (35S) promoter in order to assure high gene expression. However, the use of such promoters sometimes leads to negative effects because of the pleiotropic action of the studied gene. Precise control of gene expression could thus counteract such negative effects. This precise control could be achieved by employing synthetic promoters that induce specific expression in a specific tissue, cell type, developmental phase or growth condition.
A synthetic promoter is an artificially designed sequence. It drives the expression of a target gene, based on the presence of cis-regulatory elements or motifs, which are derived from naturally-occurring promoter elements. Since synthetic promoters only contain regulatory elements relevant for a desired expression pattern, they are transcriptionally more efficient and more compact, whereas native promoters are complex, regulated in many ways, and usually difficult to use as inducible promoters. The development of synthetic promoters could therefore help in the precise controlled expression of multiple genes involved in organ development. This project aims at developing universal synthetic promoters driving specific spatio-temporal expression during leaf development. To do so, we developed a methodology to design synthetic promoters starting from a transcriptomic analysis following Arabidopsis leaf development over time.
From the transcriptome data, co-expressed genes were selected and enriched transcription factor binding sites were identified. Next, we designed artificial promoters containing the minimal 35S promoter and 4 copies of the same motif upstream of the GUS-GFP reporter fusion. With this approach, we have validated more than 20 promoters for functionality and found 5 promoters with specific expression during leaf development. Furthermore, we are exploring the universal character by observing the expression specificity of the synthetic promoter in both maize and tomato.
With this work, we are striving to create a toolbox with universal artificial promoters marking a specific cellular process during leaf development or zone in the developing leaf.