Two IIIf Clade-bHLHs from Freesia hybrida Play Divergent Roles in Flavonoid Biosynthesis and Trichome Formation when Ectopically Expressed in Arabidopsis.

Yueqing Li,Xiang Gao,Xiaotong Shan,Shucai Wang,Ruifang Gao,Li Wang,Song Yang

doi:10.1038/srep30514

Abstract

The MBW complex, comprised by R2R3-MYB, basic helix-loop-helix (bHLH) and WD40, is a single regulatory protein complex that drives the evolution of multiple traits such as flavonoid biosynthesis and epidermal cell differentiation in plants. In this study, two IIIf Clade-bHLH regulator genes, FhGL3L and FhTT8L, were isolated and functionally characterized from Freesia hybrida. Different spatio-temporal transcription patterns were observed showing diverse correlation with anthocyanin and proanthocyanidin accumulation. When overexpressed in Arabidopsis, FhGL3L could enhance the anthocyanin accumulation through up-regulating endogenous regulators and late structural genes. Unexpectedly, trichome formation was inhibited associating with the down-regulation of AtGL2. Comparably, only the accumulation of anthocyanins and proanthocyanidins was strengthened in FhTT8L transgenic lines. Furthermore, transient expression assays demonstrated that FhGL3L interacted with AtPAP1, AtTT2 and AtGL1, while FhTT8L only showed interaction with AtPAP1 and AtTT2. In addition, similar activation of the AtDFR promoter was found between AtPAP1-FhGL3L/FhTT8L and AtPAP1- AtGL3/AtTT8 combinations. When FhGL3L was fused with a strong activation domain VP16, it could activate the AtGL2 promoter when co-transfected with AtGL1. Therefore, it can be concluded that the functionality of bHLH factors may have diverged, and a sophisticated interaction and hierarchical network might exist in the regulation of flavonoid biosynthesis and trichome formation.

Highlights

Transcription factor gene families, containing R2R3- MYB, basic helix–loop–helix and WD40 repeats (WDRs), comprise an evolutionarily conserved single regulatory protein complex, driving the evolution of multiple traits such as anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants[6,7,8]
After introduced in to Arabidopsis, divergent functionalities were observed in flavonoid biosynthesis and trichome formation, and a model that integrates the interactions of the MYB and basic helix–loop–helix (bHLH) partners within the MBW complex to explain their roles in Arabidopsis were proposed
Sequence analysis showed that the MYB interaction region (MIR) was present at the N-terminal region of the proteins; the bHLH domain involved in the formation of homo- or heterodimers with other bHLH proteins or DNA binding was located in C-terminal region

Summary

Introduction

Transcription factor gene families, containing R2R3- MYB, basic helix–loop–helix (bHLH) and WD40 repeats (WDRs), comprise an evolutionarily conserved single regulatory protein complex (designated as MBW complex), driving the evolution of multiple traits such as (pro) anthocyanin and anthocyanin biosynthesis and epidermal cell differentiation in plants[6,7,8]. Because the transcription patterns of FhTT8L were spatially and temporally consistent with anthocyanin and proanthocyanidin accumulation in flower developmental process and different plant tissues, the expression levels of endogenous genes were examined in FhTT8L transgenic lines.

Results

Conclusion