MYB-bHLH-TTG1 in a Multi-tiered Pathway Regulates Arabidopsis Seed Coat Mucilage Biosynthesis Genes Including PECTIN METHYLESTERASE INHIBITOR14 Required for Homogalacturonan Demethylesterification.

MYB-bHLH-TTG1 (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat biosynthesis pathways via a multi-tiered regulatory mechanism. The MYB genes include MYB5, MYB23 and TRANSPARENT TESTA2 (TT2), which regulate GLABRA2 (GL2), HOMEODOMAIN GLABROUS2 (HDG2) and TRANSPARENT TESTA GLABRA2 (TTG2). Here, we examine the role of PECTIN METHYLESTERASE INHIBITOR14 (PMEI14) in seed coat mucilage pectin methylesterification and provide evidence in support of multi-tiered regulation of seed coat mucilage biosynthesis genes including PMEI14. The PMEI14 promoter was active in the seed coat and developing embryo. A pmei14 mutant exhibited stronger attachment of the outer layer of seed coat mucilage, increased mucilage homogalacturonan demethylesterification and reduced seed coat radial cell wall thickness, results consistent with decreased PMEI activity giving rise to increased PME activity. Reduced mucilage release from the seeds of myb5, myb23, tt2 and gl2, hdg2, ttg2 triple mutants indicated that HDG2 and MYB23 play minor roles in seed coat mucilage deposition. Chromatin immunoprecipitation analysis found that MYB5, TT8 and seven mucilage pathway structural genes are directly regulated by MYB5. Expression levels of GL2, HDG2, TTG2 and nine mucilage biosynthesis genes including PMEI14 in the combinatorial mutant seeds indicated that these genes are positively regulated by at least two of those six TFs and that TTG1 and TTG2 are major regulators of PMEI14 expression. Our results show that MYB-bHLH-TTG1 complexes regulate mucilage biosynthesis genes, including PMEI14, both directly and indirectly via a three-tiered mechanism involving GL2, HDG2 and TTG2.

The Arabidopsis MYB5 transcription factor interacts with CASEIN KINASE2 BETA3 subunit in a yeast two-hybrid system.

Arabidopsis thaliana MYB5 collaborates with TRANSPARENT TESTA GLABRA1 (TTG1) and basic-Helix-Loop-Helix (bHLH) transcription factors to regulate seed coat, trichome and root cell differentiation. Using a yeast two-hybrid system we show that the N-terminal region of MYB5 binds directly to the serine/threonine CASEIN KINASE2 BETA3 (CK2ß3) subunit. Functions of the CASEIN KINASE2 (CK2) complex include facilitating phosphorylation of MYB transcription factors and cell cycle checkpoint regulatory proteins. Purified recombinant MYB5 protein was found to bind only weakly in vitro to the promoter of ALPHA/BETA ESTERASE/HYDROLASE4 (ABE4), a known MYB5 target gene. We propose that phosphorylation of MYB5 facilitated by the MYB5-CK2ß3 interaction enhances MYB5 binding to its target gene promoters.

MYB-bHLH-TTG1 Regulates Arabidopsis Seed Coat Biosynthesis Pathways Directly and Indirectly via Multiple Tiers of Transcription Factors.

MYB-bHLH-WDR (MBW) transcription factor (TF) complexes regulate Arabidopsis seed coat development including mucilage and tannin biosynthesis. The R2R3 MYBs MYB5, MYB23 and TRANSPARENT TESTA2 (TT2) participate in the MBW complexes with the WD-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1). These complexes regulate GLABRA2 (GL2) and TTG2 expression in developing seeds. Microarray transcriptome analysis of ttg1-1- and wild-type (Ler) developing seeds identified 246 TTG1-regulated genes, which include all known metabolic genes of the tannin biosynthetic pathway. The first detailed TTG1-dependent metabolic pathways could be proposed for the biosynthesis of mucilage, jasmonic acid (JA) and cuticle including wax ester in developing seeds. We also assigned many known and previously uncharacterized genes to the activation/inactivation of hormones, plant immunity and nutrient transport. The promoters of six cuticle pathway genes were active in developing seeds. Expression of 11 genes was determined in the developing seeds of the combinatorial mutants of MYB5, MYB23 and TT2, and in the combinatorial mutants of GL2, HOMEODOMAIN GLABROUS2 (HDG2) and TTG2. These six TFs positively co-regulated the expression of four repressor genes while three of the six TFs repressed the wax biosynthesis genes examined, suggesting that the three TFs upregulate the expression of these repressor genes, which, in turn, repress the wax biosynthesis genes. Chromatin immunoprecipitation analysis identified 21 genes directly regulated by MYB5 including GL2, HDG2, TTG2, four repressor genes and various metabolic genes. We propose a multi-tiered regulatory mechanism by which MBWs regulate tannin, mucilage, JA and cuticle biosynthetic pathways.