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

Abstract

SUMMARYWild 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.

Transcriptional control of flavonoid biosynthesis

“…2 Although the endothelium layer of the tt1 and tt16 seeds lacks PA accumulation, the chalazalmicropylar area of these mutant seeds still produces PAs. 2,3 These data suggest that PA biosynthesis at the seed base is not controlled by TT1 and TT16. TT1 is able to interact with TT2 but not with TT8 or TTG1 and ectopic expression of TT2 partially restores PA production in the tt1 and tt16 endothelial cells.…”

“…3,4,[10][11][12] Here, I summarize the current knowledge of how these novel players regulate flavonoid biosynthesis and thus plant developmental processes and further put forward a fineadjusting mechanism to complete the complex regulatory network involved in flavonoid production.…”

“…1). [2][3][4] Anthocyanin production is transcriptionally regulated by the MBW complex composed of TTG1, one R2R3-MYB protein from PRODUCTION OF ANTHOCYANIN PIGMENTS (PAP1), PAP2, MYB113, or MYB114, as well as one bHLH protein from TT8, GLABROUS3 (GL3), or ENHANCER OF GLABRA3 (EGL3) (Fig. 1).…”

“…1). [2][3][4] Several of the late flavonoid biosynthetic genes (LBGs) are activated by the MYB-bHLH-WD40 (MBW) ternary transcriptional complex comprising 3 classes of regulatory proteins, including R2R3-MYBs, bHLHs, and TRANSPARENT TESTA GLABROUS1 (TTG1; also termed WD40) (Fig. 1).…”

“…1). [2][3][4] Dihydroflavonols are then oxidized by flavonol synthase (FLS) to produce flavonols, such as quercetins and kaempferols. These early biosynthetic steps are transcriptionally regulated by the 3 closely related R2R3-MYB proteins MYB11, MYB12, and MYB111, which activate the early flavonoid biosynthetic genes CHS, CHI, F3H, and FLS1 (EBGs; Fig.…”