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.     

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.     

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.     

Arabidopsis TTG2 Regulates TRY Expression through Enhancement of Activator Complex-Triggered Activation

Trichome patterning in Arabidopsis thaliana is regulated by a regulatory feedback loop of the trichome promoting factors TRANSPARENT TESTA GLABRA1 (TTG1), GLABRA3 (GL3)/ENHANCER OF GL3 (EGL3), and GL1 and a group of homologous R3MYB proteins that act as their inhibitors. Together, they regulate the temporal and spatial expression of GL2 and TTG2, which are considered to control trichome cell differentiation. In this work, we show that TTG2 is a specific activator of TRY (but not CPC or GL2). The WRKY protein TTG2 binds to W-boxes in a minimal promoter fragment of TRY, and these W-boxes are essential for rescue of the try mutant phenotype. We further show that TTG2 alone is not able to activate TRY expression, but rather drastically enhances the activation by TTG1 and GL3. As TTG2 physically interacts with TTG1 and because TTG2 can associate with GL3 through its interaction with TTG1, we propose that TTG2 enhances the activity of TTG1 and GL3 by forming a protein complex.     

Entopically additive expression of GLABRA2 alters the frequency and spacing of trichome initiation

GLABRA2 (GL2)/ATHB-10 encodes a homeodomain protein that belongs to the homeodomain-leucine zipper family. Mutant studies have revealed that this gene is involved in trichome, root-hair and seed-coat development. We used reverse genetics to investigate the role of GL2 in trichome development. A transgene consisting of a GL2-coding fragment preceded by the cauliflower mosaic virus 35S promoter (35S::GL2) did not complement defects in the gl2-1 mutant. In the wild-type genetic background, 35S::GL2 caused gl2-mutant-like and scarcely viable phenotypes, suggesting that ectopic overexpression of GL2 interrupts endogenous GL2 function in trichome development and is toxic to plants. On the other hand, another GL2 transgene containing the GL2 promoter (pGL2::GL2) complemented the gl2-1 mutation. Entopically additive expression of GL2 by introduction of pGL2::GL2 in the wild-type genetic background noticably increased the number of trichomes and induced production of adjacent trichomes. Consistent with this result, gl2-1/+ heterozygous leaves, whose GL2 expression was expected to decrease, had fewer trichomes than +/+ leaves. These results indicate that GL2 quantitatively regulates the frequency of trichome initiation and is involved in determining trichome spacing.     

E2F and retinoblastoma related proteins may regulate GL1 expression in developing Arabidopsis trichomes

This is an addendum to our recent paper published in The Plant Journal (52:352–61). The major findings were: (1) trichomes on the leaves of gl3-sst sim double mutants developed as large multi-cellular clusters whereas wild type trichomes are composed of single cells; (2) ectopic CYCD3;1 expression in gl3-sst trichomes also resulted in trichome cluster formation; and (3) that GL1 expression is prolonged in the gl3-sst sim trichome clusters. This addendum shows that ectopic CYCD3;1 expression in gl3-sst also enhanced GL1 expression. An analysis of the GL1 promoter found two overlapping potential E2F binding sites in a region of the promoter known to be essential for GL1 function. This finding indicates that GL1 may be directly regulated by the activity of a CYCD3/CDKA complex that phosphorylates E2F-RB bound to the GL1 promoter.Key words: plant cell cycle, endoreduplication, glabra1, plant development