The Carboxyl-Terminus of TRANSPARENT TESTA GLABRA1 Is Critical for Its Functions in Arabidopsis.

Yating Wang,Wei Wang,Saddam Hussain,Hainan Tian,Kaijie Zheng,Rao Lin,Tianya Wang,Shucai Wang,Xutong Wang

doi:10.3390/ijms221810039

Abstract

The Arabidopsis WD40 repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) regulates cell fate determination, including trichome initiation and root hair formation, as well as secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production. TTG1 regulates different processes via regulating the expression of its downstream target genes by forming MYB-bHLH-WD40 (MBW) activator complexes with different R2R3 MYB and bHLH transcription factors. Here, we report the identification of the carboxyl (C)-terminus as a critical domain for TTG1′s functions in Arabidopsis. We found that the ttg1Δ15aa mutant shows pleiotropic phenotypes identical to a TTG1 loss-of-function mutant. Gene sequencing indicates that a single nucleotide substitution in TTG1 led to a premature stop at the W327 residue, leading to the production of a truncated TTG1 protein with a deletion of the last 15 C-terminal amino acids. The expression of TTG1 under the control of its native promoter fully restored the ttg1Δ15aa mutant phenotypes. Consistent with these observations, the expression levels of TTG1 downstream genes such as GLABRA2 (GL2) and CAPRICE (CPC) were reduced in the ttg1Δ15aa mutant. Assays in Arabidopsis protoplast show that TTG1Δ15aa failed to interact with the bHLH transcription factor GL3, and the deletion of the last 3 C-terminal amino acids or the 339L amino acid alone fully abolished the interaction of TTG1 with GL3. Furthermore, the expression of TTG1Δ3aa under the control of TTG1 native promoter failed to restore the ttg1Δ15aa mutant phenotypes. Taken together, our results suggest that the C-terminal domain of TTG1 is required for its proper function in Arabidopsis.

Highlights

TRANSPARENT TESTA GLABRA1 (TTG1) is a WD40 repeat protein with four WD40 motifs [1]
The available evidence suggests that, at least in the terms of regulating cell fate determination and secondary metabolism, TTG1 is able to interact with different basic helix-loop-helix transcription factors and with different R2R3 MYB transcription factors to form different MYB-bHLH-WD40 (MBW) activator complexes, thereby regulating the expression of its downstream target genes involved in cell fate determination or secondary metabolism [16,17,18,19,20]
We found that ttg1∆15aa is a TTG1 loss-of-function mutant, and assays in transfected protoplasts show that TTG1∆15aa failed to interact with GL3

Summary

Introduction

It is well known that TTG1 regulates cell fate determination including trichome and root hair formation and secondary metabolism such as flavonoid biosynthesis and seed coat mucilage production [2]. Arabidopsis loss-of-function mutants of TTG1 show a pleiotropic phenotype including glabrous leaves, increased root hairs, transparent testa seed coat, reduced anthocyanin accumulation, and seed coat mucilage production [1,3,4,5,6]. Studies in recent years have shown that TTG1 is involved in the regulation of other biological processes, such as the accumulation of fatty acids and proteins during the seed maturation [7,8], flowering time [9], as well as plant responses to biotic and abiotic stresses [10,11,12]. The available evidence suggests that, at least in the terms of regulating cell fate determination and secondary metabolism, TTG1 is able to interact with different basic helix-loop-helix (bHLH) transcription factors and with different R2R3 MYB transcription factors to form different MYB-bHLH-WD40 (MBW) activator complexes, thereby regulating the expression of its downstream target genes involved in cell fate determination or secondary metabolism [16,17,18,19,20]

Methods

Findings

Discussion

Conclusion