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.     

TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP‐type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA‐specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.     

The Arabidopsis MYB5 Transcription Factor Regulates Mucilage Synthesis,Seed Coat Development,and Trichome Morphogenesis

The Arabidopsis thaliana MYB5 gene is expressed in trichomes and seeds, including the seed coat. Constitutive expression of MYB5 resulted in the formation of more small trichomes and ectopic trichomes and a reduction in total leaf trichome numbers and branching. A myb5 mutant displayed minimal changes in trichome morphology, while a myb23 mutant produced increased numbers of small trichomes and two-branched trichomes. A myb5 myb23 double mutant developed more small rosette trichomes and two-branched trichomes than the single mutants. These results indicate that MYB5 and MYB23 regulate trichome extension and branching. The seed coat epidermal cells of myb5 and myb5 myb23 were irregular in shape, developed flattened columellae, and produced less mucilage than those of the wild type. Among the downregulated genes identified in the myb5 seeds using microarray analysis were ABE1 and ABE4 (α/β fold hydrolase/esterase genes), MYBL2, and GLABRA2. The same genes were also downregulated in transparent testa glabra1 (ttg1) seeds, suggesting that MYB5 collaborates with TTG1 in seed coat development. These genes were upregulated in leaves and roots by ectopically expressed MYB5. The MYBL2, ABE1, and ABE4 promoters were active in seeds, including seed coats, and the latter two also in trichomes. Models of the MYB5 regulatory networks involved in seed coat and trichome development are presented.     

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.     

TT8 controls its own expression in a feedback regulation involving TTG1 and homologous MYB and bHLH factors, allowing a strong and cell-specific accumulation of flavonoids in Arabidopsis thaliana

The control of TT8 expression was investigated in this study, and it was demonstrated that it constitutes a major regulatory step in the specific activation of the expression of flavonoid structural genes. First, the GUS activity generated in planta from a TT8::uidA construct revealed cell-specific activation of the TT8 promoter consistent with the known involvement of the TT8 bHLH factor in proanthocyanidin, anthocyanin and mucilage biosynthesis. Moreover, the activity of this reporter construct was strongly affected in ttg1, TT2 overexpressers (OE), and PAP1-OE, suggesting interplay between TT2, PAP1, TTG1 and the activation of the TT8 promoter in planta. To further investigate the mechanisms involved, we used 35S::TT2-GR and 35S::TTG1-GR transgenic plants (expressing fusion proteins with the glucocorticoid receptor), as well as one-hybrid experiments, to determine the direct effect of these factors on TT8 expression. Interestingly, in vivo binding of TT2 and PAP1 to the TT8 promoter was dependent on the simultaneous expression of TT8 or the homologous bHLH factors GL3 and EGL3. Consistent with these results, the activity of the TT8::uidA reporter was strongly affected in the seed endothelium of a tt8 mutant. Similarly, a strong decrease in the level of TT8 mRNA was detected in the siliques of a gl3 x egl3 mutant and in plants that express a dominant negative form of the PAP1 protein, suggesting that TT8 expression is controlled by different combinations of MYB and bHLH factors in planta. The importance of this positive feedback mechanism in the strong and specific induction of proanthocyanidin biosynthesis in the seed coat of Arabidopsis thaliana is discussed.