TTG2 controls the developmental regulation of seed coat tannins in Arabidopsis by regulating vacuolar transport steps in the proanthocyanidin pathway

Abstract

The brown color of Arabidopsis seeds is caused by the deposition of proanthocyanidins (PAs or condensed tannins) in their inner testa layer. A transcription factor complex consisting of TT2, TT8 and TTG1 controls expression of PA biosynthetic genes, just as similar TTG1-dependent complexes have been shown to control flavonoid pigment pathway gene expression in general. However, PA synthesis is controlled by at least one other gene. TTG2 mutants lack the pigmentation found in wild-type seeds, but produce other flavonoid compounds, such as anthocyanins in the shoot, suggesting that TTG2 regulates genes in the PA biosynthetic branch of the flavonoid pathway. We analyzed the expression of PA biosynthetic genes within the developing seeds of ttg2-1 and wild-type plants for potential TTG2 regulatory targets. We found that expression of TT12, encoding a MATE type transporter, is dependent on TTG2 and that TTG2 can bind to the upstream regulatory region of TT12 suggesting that TTG2 directly regulates TT12. Ectopic expression of TT12 in ttg2-1 plants partially restores seed coat pigmentation. Moreover, we show that TTG2 regulation of TT12 is dependent on TTG1 and that TTG1 and TTG2 physically interact. The observation that TTG1 interacts with TTG2, a WRKY type transcription factor, proposes the existence of a novel TTG1-containing complex, and an addendum to the existing paradigm of flavonoid pathway regulation.

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

“…However, a remarkable number of WRKY proteins have also important roles in development and in the control of secondary metabolic pathways (Schluttenhofer and Yuan, 2015). This is the case of the well-characterized TTG2 of Arabidopsis that is a regulator of seed and trichome development (Johnson et al, 2002; Garcia et al, 2005; Ishida et al, 2007; Gonzalez et al, 2016). Two WRKY factors extremely close to AtTTG2 have recently been characterized, PH3 of P. hybrida , a novel regulator of vacuolar acidification in petals (Verweij et al, 2016), and TTG2 of Brassica napus involved in response to salt stress and in trichome development (Li et al, 2015).…”

“…Moreover, both AtTTG2 and PhPH3 are part of a similar regulative and transcriptional network, in which the respective MBW complexes activate their expression and also require their interaction to activate the downstream pathways (Gonzalez et al, 2016; Verweij et al, 2016). In this study we provide evidence that VvWRKY26 is able to fulfill the functions of PhPH3 when expressed in petunia ph3 mutant, and functional analogies were also found between VvWRKY26 and the homolog AtTTG2 regarding proanthocyanidin biosynthesis as indicated by the induction of numerous PA-related genes in Sultana plants over-expressing VvWRKY26 .…”

“…The Arabidopsis homolog TTG2 was also shown to be a key regulator of PA accumulation, and ttg2 mutants have a reduced deposition of PAs in the seed coat. AtTTG2 drives the PA vacuolar storage by directly regulating the ATPase proton pump TT13 and the MATE vacuolar transporter TT12 (Appelhagen et al, 2015; Gonzalez et al, 2016). …”

“…Similarly to PhPH3, AtTTG2 interacts with the MBW complex for the control of the vacuolar transport of PAs during seed coat development by regulating the expression of AtTT13 and also by directly activating the expression of TRANSPARENT TESTA 12 ( AtTT12 ), a MATE transporter of glycosilated epicathechin PA precursors (Debeaujon et al, 2001; Gonzalez et al, 2016). Another functional homolog of AtTTG2 was identified in Brassica napus .…”

A renaissance in plant development

“…While the biochemical function of Mediator is highly conserved across eukaryotes, the extent to which different Mediator subunits have specific roles in particular plant developmental processes, versus a more general role in transcriptional control, is still an open question. The intricacies of transcriptional control are also addressed in Gonzalez, et al, ( 2016) who identify novel transcriptional complexes involved in flavonoid pathway regulation and seed coat differentiation.…”