Roles of the GLABROUS1 and TRANSPARENT TESTA GLABRA Genes in Arabidopsis Trichome Development

Arabidopsis trichomes are branched, single-celled epidermal hairs. These specialized cells provide a convenient model for investigating the specification of cell fate in plants. Two key genes regulating the initiation of trichome development are GLABROUS1 (GL1) and TRANSPARENT TESTA GLABRA (TTG). GL1 is a member of the myb gene family. The maize R gene, which can functionally complement the Arabidopsis ttg mutation, encodes a basic helix-loop-helix protein. We used constitutively expressed copies of the GL1 and R genes to test hypotheses about the roles of GL1 and TTG in trichome development. The results support the hypothesis that TTG and GL1 cooperate at the same point in the trichome developmental pathway. Furthermore, the constitutive expression of both GL1 and R in the same plant caused trichomes to develop on all shoot epidermal surfaces. Results were also obtained indicating that TTG plays an additional role in inhibiting neighboring cells from becoming trichomes.     

GL3 encodes a bHLH protein that regulates trichome development in arabidopsis through interaction with GL1 and TTG1

Arabidopsis trichome development and differentiation is a well-studied model for plant cell-fate determination and morphogenesis. Mutations in TRANSPARENT TESTA GLABRA1 (TTG1) result in several pleiotropic defects including an almost complete lack of trichomes. The complex phenotype caused by ttg1 mutations is suppressed by ectopic expression of the maize anthocyanin regulator R. Here it is demonstrated that the Arabidopsis trichome development locus GLABRA3 (GL3) encodes an R homolog. GL3 and GLABRA1 (GL1) interact when overexpressed together in plants. Yeast two-hybrid assays indicate that GL3 participates in physical interactions with GL1, TTG1, and itself, but that GL1 and TTG1 do not interact. These data suggest a reiterated combinatorial model for the differential regulation of such diverse developmental pathways as trichome cell-fate determination, root hair spacing, and anthocyanin secondary metabolism.     

Mutations in the pale aleurone color1 regulatory gene of the Zea mays anthocyanin pathway have distinct phenotypes relative to the functionally similar TRANSPARENT TESTA GLABRA1 gene in Arabidopsis thaliana

The pale aleurone color1 (pac1) locus, required for anthocyanin pigment in the aleurone and scutellum of the Zea mays (maize) seed, was cloned using Mutator transposon tagging. pac1 encodes a WD40 repeat protein closely related to anthocyanin regulatory proteins ANTHOCYANIN11 (AN11) (Petunia hybrida [petunia]) and TRANSPARENT TESTA GLABRA1 (TTG1) (Arabidopsis thaliana). Introduction of a 35S-Pac1 transgene into A. thaliana complemented multiple ttg1 mutant phenotypes, including ones nonexistent in Z. mays. Hybridization of Z. mays genomic BAC clones with the pac1 sequence identified an additional related gene, mp1. PAC1 and MP1 deduced protein sequences were used as queries to build a phylogenetic tree of homologous WD40 repeat proteins, revealing an ancestral gene duplication leading to two clades in plants, the PAC1 clade and the MP1 clade. Subsequent duplications within each clade have led to additional WD40 repeat proteins in particular species, with all mutants defective in anthocyanin expression contained in the PAC1 clade. Substantial differences in pac1, an11, and ttg1 mutant phenotypes suggest the evolutionary divergence of regulatory mechanisms for several traits that cannot be ascribed solely to divergence of the dicot and monocot protein sequences.     

Two WD-repeat genes from cotton are functional homologues of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis><Emphasis Type="Italic">TRANSPARENT TESTA GLABRA1</Emphasis> (<Emphasis Type="Italic">TTG1</Emphasis>) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsis thaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.     

TT2, TT8, and TTG1 synergistically specify the expression of BANYULS and proanthocyanidin biosynthesis in Arabidopsis thaliana

Genetic analyses have demonstrated that together with TTG1, a WD-repeat (WDR) protein, TT2 (MYB), and TT8 (bHLH) are necessary for the correct expression of BANYULS (BAN). This gene codes for the core enzyme of proanthocyanidin biosynthesis in Arabidopsis thaliana seed coat. The interplays of TT2, TT8, and their closest MYB/bHLH relatives, with TTG1 and the BAN promoter have been investigated using a combination of genetic and molecular approaches, both in yeast and in planta. The results obtained using glucocorticoid receptor fusion proteins in planta strongly suggest that TT2, TT8, and TTG1 can directly activate BAN expression. Experiments using yeast two- and three-hybrid clearly demonstrated that TT2, TT8, and TTG1 can form a stable ternary complex. Furthermore, although TT2 and TT8 were able to bind to the BAN promoter when simultaneously expressed in yeast, the activity of the complex correlated with the level of TTG1 expression in A. thaliana protoplasts. In addition, transient expression experiments revealed that TTG1 acts mainly through the bHLH partner (i.e. TT8 or related proteins) and that TT2 cannot be replaced by any other related A. thaliana MYB proteins to activate BAN. Finally and consistent with these results, the ectopic expression of TT2 was sufficient to trigger BAN activation in vegetative parts, but only where TTG1 was expressed. Taken together, these results indicate that TT2, TT8, and TTG1 can form a ternary complex directly regulating BAN expression in planta.