The Basic Helix-Loop-Helix Transcription Factor SmbHLH1 Represses Anthocyanin Biosynthesis in Eggplant.

Zhaofei Duan,Jing Li,Guobin Yang,Fengjuan Yang,Shiyu Tian,Min Wei

doi:10.3389/fpls.2021.757936

Abstract

Many basic helix-loop-helix transcription factors (TFs) have been reported to promote anthocyanin biosynthesis in numerous plant species, but little is known about bHLH TFs that inhibit anthocyanin accumulation. In this study, SmbHLH1 from Solanum melongena was identified as a negative regulator of anthocyanin biosynthesis. However, SmbHLH1 showed high identity with SmTT8, which acts as a SmMYB113-dependent positive regulator of anthocyanin-biosynthesis in plants. Overexpression of SmbHLH1 in eggplant caused a dramatic decrease in anthocyanin accumulation. Only the amino acid sequences at the N and C termini of SmbHLH1 differed from the SmTT8 sequence. Expression analysis revealed that the expression pattern of SmbHLH1 was opposite to that of anthocyanin accumulation. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that SmbHLH1 could not interact with SmMYB113. Dual-luciferase assay demonstrated that SmbHLH1 directly repressed the expression of SmDFR and SmANS. Our results demonstrate that the biological function of bHLHs in anthocyanin biosynthesis may have evolved and provide new insight into the molecular functions of orthologous genes from different plant species.

Highlights

Anthocyanins are water-soluble pigments that are responsible for the red, purple, and blue colors of different organs in a wide range of plants (Mol et al, 1998; Koes et al, 2005; Allan et al, 2008)
A phylogenetic tree was constructed to analyze the relationships of SmbHLH1 and SmTT8 with all bHLH proteins from Arabidopsis, Solanum lycopersicum, Malus × domestica, Vitis vinifera, Fragaria × ananassa, Petunia × hybrida, Prunus persica, Chimonanthus praecox, and Medicago truncatula known to be involved in the anthocyanin biosynthesis pathway (Supplementary Figure S3; Li et al, 2017; Zhao et al, 2020)
We found that SmbHLH1, SmTT8, and MdbHLH3 were clustered into one clade by phylogenetic analysis (Supplementary Figure S3), and the N terminus and C terminus of SmbHLH1 differed significantly from those of SmTT8 (Figure 1)

Summary

Introduction

Anthocyanins are water-soluble pigments that are responsible for the red, purple, and blue colors of different organs in a wide range of plants (Mol et al, 1998; Koes et al, 2005; Allan et al, 2008). Function of SmbHLH1 in Eggplant isomerase (CHI), flavanone-3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′5′-hydroxylase (F3′5′H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and a 3-glucosyltransferase (3GT; Koes et al, 2005). These anthocyanin biosynthetic genes are transcriptionally regulated by a MYB-bHLH-WD40 (MBW) complex, the crucial role of which has been well-documented across many plants, such as Arabidopsis, apple, tomato, and so on (Xie et al, 2012; Tohge et al, 2017; Sun et al, 2020a). MdbHLH3 interacts with MdMYB1, MdMYB9, and MdMYB11 to promote anthocyanin or proanthocyanidin (PA) accumulation (Xie et al, 2012; An et al, 2014)

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Conclusion