Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls endoreduplication and flowering development in addition to trichome and root hair formation

CAPRICE (CPC) encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis thaliana. Three additional CPC-like MYB genes, TRY (TRIPTYCHON), ETC1 (ENHANCER OF TRY AND CPC 1) and ETC2 (ENHANCER OF TRY AND CPC 2) act in a redundant manner with CPC in trichome and root hair patterning. In this study, we identified an additional homolog, CPC-LIKE MYB 3 (CPL3), which has high sequence similarity to CPC, TRY, ETC1 and ETC2. Overexpression of CPL3 results in the suppression of trichomes and overproduction of root hairs, as has been observed for CPC, TRY, ETC1 and ETC2. Morphological studies with double, triple and quadruple homolog mutants indicate that the CPL3 gene cooperatively regulates epidermal cell differentiation with other CPC homologs. Promoter-GUS analyses indicate that CPL3 is specifically expressed in leaf epidermal cells, including stomate guard cells. Notably, the CPL3 gene has pleiotropic effects on flowering development, epidermal cell size and trichome branching through the regulation of endoreduplication.     

Analysis of IIId, IIIe and IVa group basic-helix-loop-helix proteins expressed in Arabidopsis root epidermis

Differentiation of Arabidopsis epidermal cells into root hairs and trichomes is a functional model system for understanding plant cell development. Previous studies showed that one of the Arabidopsis basic-helix-loop-helix (AtbHLH) proteins, GLABRA3 (GL3), is involved in root-hair and trichome differentiation. We analyzed 11 additional AtbHLH genes with homology to GL3. Estimation of the phylogeny based on amino acid sequences of the bHLH region suggests that 11 AtbHLH genes used in this study evolved by duplications of a single common GL3 ancestor. Promoter-GUS analysis showed that AtbHLH006, AtbHLH013, AtbHLH017 and AtbHLH020 were expressed in roots. Among them, AtbHLH006 and AtbHLH020 were preferentially expressed in root epidermal non-hair cells. Consistent with the expression patterns from promoter-GUS analysis, GFP fluorescence was observed in the nuclei of root epidermal non-hair cells of AtbHLH006p::AtbHLH006:GFP and AtbHLH020p::AtbHLH020:GFP transgenic plants. However, AtbHLH006 and AtbHLH0020 proteins did not interact with epidermis-specific MYB proteins and TTG1. Taken together, AtbHLH006 and AtbHLH020 may function in root epidermal cells, but other GL3-like bHLH proteins may have evolved to regulate different processes.     

Root hair formation at the root-hypocotyl junction in CPC-LIKE MYB double and triple mutants of Arabidopsis

In Arabidopsis thaliana, R3-type MYB genes, CAPRICE (CPC) and its family of genes including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and CPC-LIKE MYB3 cooperatively regulate epidermal cell differentiation. Root hair formation is greatly reduced by a mutation in CPC, and try and etc1 enhance this phenotype. In this study, we demonstrate that CPC, TRY and ETC1 are also involved in root hair formation at the root-hypocotyl junction. The cpc try and cpc etc1 double mutants showed a reduced number of root hairs in that area. Additionally, the expression of ETC1::GUS was higher near this area. These results suggest that CPC family of genes also cooperatively regulates root hair formation at the root-hypocotyl junction in unique ways.     

A network of redundant bHLH proteins functions in all TTG1-dependent pathways of Arabidopsis

GLABRA3 (GL3) encodes a bHLH protein that interacts with the WD repeat protein, TTG1. GL3 overexpression suppresses the trichome defect of the pleiotropic ttg1 mutations. However, single gl3 mutations only affect the trichome pathway with a modest trichome number reduction. A novel unlinked bHLH-encoding locus is described here, ENHANCER OF GLABRA3 (EGL3). When mutated, egl3 gives totally glabrous plants only in the gl3 mutant background. The double bHLH mutant, gl3 egl3, has a pleiotropic phenotype like ttg1 having defective anthocyanin production, seed coat mucilage production, and position-dependent root hair spacing. Furthermore, the triple bHLH mutant, gl3 egl3 tt8, phenocopies the ttg1 mutation. Yeast two-hybrid and plant overexpression studies show that EGL3, like GL3, interacts with TTG1, the myb proteins GL1, PAP1 and 2, CPC and TRY, and it will form heterodimers with GL3. These results suggest a combinatorial model for TTG1-dependent pathway regulation by this trio of partially functionally redundant bHLH proteins.     

Distinct and overlapping roles of single-repeat MYB genes in root epidermal patterning

Cell specification in the root epidermis of Arabidopsis generates a position-dependent pattern of root-hair cells and non-hair cells. Here we conduct a comprehensive analysis of the five members of a single-repeat R3 MYB gene family, including CAPRICE (CPC), TRIPTYCHON (TRY), ENHANCER of TRY and CPC 1, 2, and 3 (ETC1, ETC2, and ETC3), and study their role and functional relationship in root epidermal cell specification. Based on genetic and expression analyses, CPC, TRY and ETC1, but not ETC2 or ETC3, promote the hair cell fate by inhibiting non-hair specification. Further, we find that single-repeat MYB activity is required for epidermal patterning throughout root development, beginning during embryogenesis. We also identify a novel regulatory interaction whereby GLABRA2 (GL2) promotes TRY (but not CPC or ETC1) expression in the root epidermis, which generates a second lateral inhibition feedback loop. Gene fusion experiments combining CPC regulatory elements with protein-coding regions of each single-repeat MYB gene suggest that all five proteins are functionally similar, although TRY and ETC2 exhibit distinctions from CPC/ETC1/ETC3. These results provide new insight into the function of these single-repeat MYBs and suggest that divergence of their regulatory sequences is largely responsible for their distinct roles in epidermal cell patterning.