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

Abstract

SummaryGenetic 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.

Sugar-Hormone Cross-Talk in Anthocyanin Biosynthesis

“…Transcription factors from the R2R3-MYB, bHLH and WD40 classes interact to form MBW regulatory complexes (Borevitz et al, 2000;Gonzalez et al, 2008;Tohge et al, 2005;Zhang et al, 2003). For instance, TRANSPARENT TESTA 2 (TT2; MYB123) requires the cofactors TT8 (bHLH42) and TTG1 (WD40-repeat protein) to form a ternary MBW complex that regulates the expression of the DFR and ANR genes, which are involved in the accumulation of proanthocyanidins in the seed coat (Baudry et al, 2004;Nesi et al, 2000;. Similarly, MYB75/PAP1 (PRODUCTION OF ANTHOCYANIN PIGMENT1) and MYB90/PAP2, which are members of the R2R3-MYB family, are responsible for bHLH co-factor-dependent transcriptional activation of phenylalanine-ammonia lyase (PAL), CHS, DFR and glutathione-S-transferase (GST), which are key enzymes of the anthocyanin biosynthesis pathway (Hichri et al, 2011).…”

Arabidopsis TRANSPARENT TESTA GLABRA2Is Directly Regulated by R2R3 MYB Transcription Factors and Is Involved in Regulation ofGLABRA2Transcription in Epidermal Differentiation

“…In trichomes, GL1-GL3/EGL3-TTG1 complexes should promote TTG2 expression, as shown by the binding of GL1 to the MST region in yeast assays (Payne et al, 2000). In seed coats, TT2-TT8-TTG1 complexes should promote TTG2 expression, because TT2 protein also binds the MST region in yeast assays (Baudry et al, 2004). TTG2 expression in leaf blades was repressed in the egl3, gl3 egl3, and ttg1 mutants (Johnson et al, 2002).…”

Endosperm and Nucellus Develop Antagonistically in Arabidopsis Seeds

“…For complementation analyses, the 3.4ProTT16:gTT16, 1.6ProTT16:gTT16, and ProTT1:gTT16 clones were recombined into the pGWB1 binary vector (Nakagawa et al, 2007). For GR-inducible analyses, the TT16 genomic sequence was recombined into the pR1R2DGR binary vector (Baudry et al, 2004). For overexpression analyses, the HVA22D coding sequence was recombined into the pMDC32 binary vector (Curtis and Grossniklaus, 2003).…”