Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

Abstract

The WD40 proteins ANTHOCYANIN11 (AN11) from petunia (Petunia hybrida) and TRANSPARENT TESTA GLABRA1 (TTG1) from Arabidopsis thaliana and associated basic helix-loop-helix (bHLH) and MYB transcription factors activate a variety of differentiation processes. In petunia petals, AN11 and the bHLH protein AN1 activate, together with the MYB protein AN2, anthocyanin biosynthesis and, together with the MYB protein PH4, distinct genes, such as PH1 and PH5, that acidify the vacuole. To understand how AN1 and AN11 activate anthocyanin biosynthetic and PH genes independently, we isolated PH3. We found that PH3 is a target gene of the AN11-AN1-PH4 complex and encodes a WRKY protein that can bind to AN11 and is required, in a feed-forward loop, together with AN11-AN1-PH4 for transcription of PH5. PH3 is highly similar to TTG2, which regulates hair development, tannin accumulation, and mucilage production in Arabidopsis. Like PH3, TTG2 can bind to petunia AN11 and the Arabidopsis homolog TTG1, complement ph3 in petunia, and reactivate the PH3 target gene PH5. Our findings show that the specificity of WD40-bHLH-MYB complexes is in part determined by interacting proteins, such as PH3 and TTG2, and reveal an unanticipated similarity in the regulatory circuitry that controls petunia vacuolar acidification and Arabidopsis hair development. INTRODUCTIONRegulatory complexes consisting of a MYB, basic helix-loop-helix (bHLH), and WD40 protein (MBW complexes) control multiple pathways involved in the differentiation of epidermal cells in angiosperms (reviewed in Broun, 2005;Koes et al., 2005;Ramsay and Glover, 2005;Ishida et al., 2008). The best understood function of this complex involves the biosynthesis of anthocyanin pigments that color many flowers and fruits. Closely related, and in most cases functionally exchangeable, MYB, bHLH, and WD40 proteins collaboratively activate the transcription of genes that encode enzymes of the anthocyanin pathway in a broad range of species, including distantly related dicots like Arabidopsis thaliana (a Rosid) and petunia (Petunia hybrida; an Asterid) and monocots like maize (Zea mays; Koes et al., 2005;Hichri et al., 2011;Petroni and Tonelli, 2011). In addition, related MBW complexes activate numerous other pathways and processes, such as vacuolar acidification, formation of seed mucilage, and the development of hair cells, a process that seems restricted to smaller sets of species (Koes et al., 2005;Ramsay and Glover, 2005;Serna and Martin, 2006).In petunia flowers, the WD40 protein ANTHOCYANIN11 (AN11) and the bHLH factor AN1 activate structural anthocyanin genes in floral tissues, such as petals and anthers, and a few vegetative tissues (Quattrocchio et al., 1993;de Vetten et al., 1997;Spelt et al., 2000). To this end, AN11 and AN1 interact with paralogous and exchangeable MYB proteins, which are encoded by AN2, AN4, and DEEP PURPLE and are expressed in different tissues (Quattrocchio et al., 1999(Quattrocchio et al., , 2006Albert et al., 2011). Furthermore, AN1 and AN11 activate, in concert...

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

“…To identify the grapevine WRKY sequence homolog to PhPH3, the predicted proteome of the near-homozygous PN40024 genotype of Vitis vinifera “Pinot noir” 12X V1 version (http://genomes.cribi.unipd.it) was queried using the conserved amino acid sequence of PhPH3 (Verweij et al, 2016). …”

“…The WRKY transcription factor PhPH3 has recently been characterized in petunia as an additional key regulator of vacuolar acidification, acting downstream and in concert with the MBW complex and controlling the expression of PhPH1 and PhPH5 (Verweij et al, 2016). The ph3 mutants show an increase of petal homogenate pH that causes a shift in petal color, an abnormal seed development and female sterility (Verweij et al, 2016).…”

“…The ph3 mutants show an increase of petal homogenate pH that causes a shift in petal color, an abnormal seed development and female sterility (Verweij et al, 2016). …”

“…The WRKY proteins are classified in groups I–III based on both the number of WRKY domains and the features of their zinc-finger motif (Eulgem et al, 2000). PhPH3, AtTTG2 and BnTTG2 proteins are all members of group Ia with two WRKY domains and a C2H2 zinc-finger motif (Johnson et al, 2002; Xie et al, 2005; Li et al, 2015; Verweij et al, 2016). …”

Chemical analysis of color change and transcriptional profile in flavonoids biosynthesis during flower development of Tibouchina pulchra (Cham.) Cong.

“…However, when the flowers open, the petal cell pH increases up to 7.7, and the pigment changes color to sky blue (Yoshida et al 1995). Recently, studying the genetic determinants of P. hybrida flower pigmentation, Verweij et al (2016) have identified PH3…”