Identification of a new R3 MYB type repressor and functional characterization of the members of the MBW transcriptional complex involved in anthocyanin biosynthesis in eggplant (S. melongena L.).

Moglia Andrea,Guerrieri Alessandra,Anna Milani Maria,Elia Florio Francesco,Marengo Arianna,Leonardo Rotino Giuseppe,Bassolino Laura,Cagliero Cecilia,Rubiolo Patrizia,Toppino Laura,Iacopino Sergio,Lanteri Sergio,Barchi Lorenzo,Comino Cinzia,Serena Aceto

doi:10.1371/journal.pone.0232986

Abstract

Here we focus on the highly conserved MYB-bHLH-WD repeat (MBW) transcriptional complex model in eggplant, which is pivotal in the transcriptional regulation of the anthocyanin biosynthetic pathway. Through a genome-wide approach performed on the recently released Eggplant Genome (cv. 67/3) previously identified, and reconfirmed by us, members belonging to the MBW complex (SmelANT1, SmelAN2, SmelJAF13, SmelAN1) were functionally characterized. Furthermore, a regulatory R3 MYB type repressor (SmelMYBL1), never reported before, was identified and characterized as well.Through a qPCR approach, we revealed specific transcriptional patterns of candidate genes in different plant tissue/organs at two stages of fruit development. Two strategies were adopted for investigating the interactions of bHLH partners (SmelAN1, SmelJAF13) with MYB counterparts (SmelANT1, SmelAN2 and SmelMYBL1): Yeast Two Hybrid (Y2H) and Bimolecular Fluorescent Complementation (BiFC) in A. thaliana mesophylls protoplast. Agro-infiltration experiments highlighted that N. benthamiana leaves transiently expressing SmelANT1 and SmelAN2 showed an anthocyanin-pigmented phenotype, while their co-expression with SmelMYBL1 prevented anthocyanin accumulation. Our results suggest that SmelMYBL1 may inhibits the MBW complex via the competition with MYB activators for bHLH binding site, although this hypothesis requires further elucidation.

Highlights

Anthocyanins are the major plant flavonoid compounds, which confer appealing colours to flowers and fruits and contribute to stress tolerance [1,2]
The bHLH encoding genes were spotted by screening the genome of S. melongena cv. 67/3 [12] with 13 known plant proteins related to anthocyanin synthesis belonging to the subgroup IIIf [35] (S1 File), whose members are known to be involved in flavonoid regulations
The eggplant bHLHs were used to construct a phylogenetic tree based on the NJ method (Fig 1) together with 13 known plant proteins related to anthocyanin synthesis belonging to the subgroup IIIf

Summary

Introduction

Anthocyanins are the major plant flavonoid compounds, which confer appealing colours to flowers and fruits and contribute to stress tolerance [1,2]. The anthocyanin pathway represents one branch of flavonoid metabolism and it is a very conserved network in many plant species, with most of the genes encoding for enzymes and regulatory transcription factors (TFs) identified in several plant species [5]. The anthocyanin pathway is one of the most finely tuned and it is under the control of Early (EBGs) and Late (LBGs) Biosynthetic Genes in dicotyledonous species [6,7]. Chalcone synthase (CHS), Chalcone-flavonone isomerase (CHI), Flavanone 3-hydroxylase (F3H) and Flavonol synthase (FLS) are common EBGs involved in the biosynthesis of all downstream flavonoids, whose enzymatic steps are controlled by co-activator independent and functionally redundant ‘R2R3-type MYB’ regulatory genes (MYB11, MYB12, MYB111) [8]

Methods

Results

Conclusion