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

TLDR

Genetic studies show that TT2, TT8, and the WD‑repeat protein TTG1 are required for expression of BANYULS, the core enzyme of proanthocyanidin biosynthesis in Arabidopsis seed coat. The authors investigated the interactions of TT2, TT8, and their closest MYB/bHLH relatives with TTG1 and the BAN promoter using genetic, yeast, and plant‑based assays. TT2, TT8, and TTG1 form a stable ternary complex that directly activates BANYULS expression, with activity dependent on TTG1 levels and requiring TT2; ectopic TT2 activates BAN only in TTG1‑expressing tissues, confirming the complex’s direct regulatory role in planta.

Abstract

Summary 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 .