Understanding the cellular differentiation of multicellular organisms requires the characterization of genes whose expression is modulated in a cell type-specific manner. The Arabidopsis (Arabidopsis thaliana) root hair cell is one model for studying cellular differentiation. In this study, root hair cell-specific genes were screened by a series of in silico and experimental filtration procedures. This process included genome-wide screening for genes with a root hair-specific cis-element in their promoters, filtering root-specific genes from the root hair-specific cis-element-containing genes, further filtering of genes that were suppressed in root hair-defective plant lines, and experimental confirmation by promoter assay. These procedures revealed 19 root hair-specific genes, including many protein kinases and cell wall-related genes, most of which have not been characterized thus far. Functional analyses of these root hair-specific genes with loss-of-function mutants and overexpressing transformants revealed that they play roles in hair growth and morphogenesis. This study demonstrates that a defined cis-element can serve as a filter to screen certain cell type-specific genes and implicates many new root hair-specific genes in root hair development.

Vascular plants develop distinctive root hair distribution patterns in the root epidermis, depending on the taxon. The three patterns, random (Type 1), asymmetrical cell division (Type 2), and positionally cued (Type 3), are controlled by different upstream fate-determining factors that mediate expression of root hair cell-specific genes for hair morphogenesis. Here, we address whether these root hair genes possess a common transcriptional regulatory module (cis-element) determining celltype specificity despite differences in the final root hair pattern. We identified Arabidopsis thaliana expansinA7 (At EXPA7) orthologous (and paralogous) genes from diverse angiosperm species with different hair distribution patterns. The promoters of these genes contain conserved root hair-specific cis-elements (RHEs) that were functionally verified in the Type-3 Arabidopsis root. The promoter of At EXPA7 (Type-3 pattern) also showed hair cell-specific expression in the Type 2 rice (Oryza sativa) root. Root hair-specific genes other than EXPAs also carry functionally homologous RHEs in their promoters. The RHE core consensus was established by a multiple alignment of functionally characterized RHEs from different species and by highresolution analysis of At EXPA7 RHE1. Our results suggest that this regulatory module of root hair-specific genes has been conserved across angiosperms despite the divergence of upstream fate-determining machinery.

Cell differentiation ultimately relies on the regulation of cell type-specific genes. For a root hair cell to undergo morphogenesis, diverse cellular processes including cell-wall loosening must occur in a root hair cell-specific manner. Previously, we identified and characterized root hairspecific cis-elements (RHE) from the genes encoding the cell wall-loosening protein EXPANSIN A (EXPA) which functions preferentially on dicot cell walls. This study reports two root hair-specific grass EXPB genes that contain RHEs. These genes are thought to encode proteins that function more efficiently on grass cell walls. The proximal promoter regions of two orthologous EXPB genes from rice (Oryza sativa; OsEXPB5) and barley (Hordeum vulgare; HvEXPB1) included RHE motifs. These promoters could direct root hair-specific expression of green fluorescent protein (GFP) in the roots of rice and Arabidopsis (Arabidopsis thaliana). Promoter deletion analyses demonstrated that the RHE motifs are necessary for root hairspecific expression of these EXPB promoters. Phylogenetic analysis of EXP protein sequences indicated that grass EXPBs are the only orthologs to these root hair-specific EXPBs, separating dicot EXPBs to distal branches of the tree. These results suggest that RHE-containing root hair-specific EXPB genes have evolved for grass-specific cell wall modification during root hair morphogenesis.