Promoter analysis reveals conserved 30-base-pair core sequences controlling the nonhair-cell preferential expression of WEREWOLF in Arabidopsis

The root epidermis of Arabidopsis (Arabidopsis thaliana) consists of two distinct cell types: hair (H) cells and non-hair (N) cells, whose patterning is regulated by a network of genes. Among these, the WEREWOLF (WER) gene, encoding an R2R3 MYB transcription factor, acts as a master regulator by promoting the expression of key downstream genes, such as GLABRA2 and CAPRICE. However, the mechanisms controlling WER expression have remained largely unexplored. In this study, we analyzed WER promoter to identify putative cis-regulatory elements that govern its N-position-preferential expression. We generated a series of WER promoter constructs with progressive 5' truncations, internal deletions/substitutions, and synthetic 18x tandem repeats of core elements, each driving expression of a β-glucuronidase (GUS) reporter gene in the presence of the nos terminator. We discovered that the region between -420 and -346 bp was required for the N-position-preferential expression. When synthetic 18x core elements from this region were used to drive GUS expression with WER terminator, the -420 to -391 bp and -390 to -361 bp cores showed weak N-position expression in the root epidermis, while the -375 to -346 bp core displayed moderate N-position expression. Additionally, WER expression driven by the -420 to -391 bp, -390 to -361 bp, and -375 to -346 bp 18x cores successfully complemented the wer mutant phenotype. These findings suggest that the cis-regulatory elements responsible for N-position-preferential expression are dispersed across the WER promoter, and the -375 to -346 bp region plays a major role in driving N-position-preferential expression. In addition, our results indicate that WER terminator is required for the proper functioning of these cis-regulatory elements.