NtbHLH1, a JAF13-like bHLH, interacts with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus

Yuxin Fan,Andrew C Allan,Ping Zhou,Lihui Zeng,Jiayu Peng,Jiacheng Wu,Ruijie He

doi:10.1186/s12870-021-03050-1

Abstract

BackgroundFlavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. However, in Chinese Narcissus (Narcissus tazetta L. var. chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear.ResultsIn this work, genes related to the regulatory complex, NtbHLH1 and a R2R3-MYB NtMYB6, were cloned from Chinese Narcissus. Phylogenetic analysis indicated that NtbHLH1 belongs to the JAF13 clade of bHLH IIIf subgroup, while NtMYB6 was highly homologous to positive regulators of proanthocyanidin biosynthesis. Both NtbHLH1 and NtMYB6 have highest expression levels in basal plates of Narcissus, where there is an accumulation of proanthocyanidin. Ectopic over expression of NtbHLH1 in tobacco resulted in an increase in anthocyanin accumulation in flowers, and an up-regulation of expression of the endogenous tobacco bHLH AN1 and flavonoid biosynthesis genes. In contrast, the expression level of LAR gene was significantly increased in NtMYB6-transgenic tobacco. Dual luciferase assays showed that co-infiltration of NtbHLH1 and NtMYB6 significantly activated the promoter of Chinese Narcissus DFR gene. Furthermore, a yeast two-hybrid assay confirmed that NtbHLH1 interacts with NtMYB6.ConclusionsOur results suggest that NtbHLH1 may function as a regulatory partner by interacting directly with NtMYB6 to enhance proanthocyanidin accumulation in Chinese Narcissus.

Highlights

Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway
Our results suggest that NtbHLH1 plays a role in flavonoid biosynthesis and interacts with NtMYB6 to directly promote proanthocyanidin biosynthesis in Chinese narcissus
The 3 ́ end sequence of NtbHLH1 was obtained by 3 ́ RACE and the full length of open reading frame (ORF) was cloned by Real-time PCR (RT-PCR) (Supplemental Fig. 1) (GenBank number QDS02912.1)

Summary

Introduction

Flavonoid biosynthesis in plants is primarily regulated at the transcriptional level by transcription factors modulating the expression of genes encoding enzymes in the flavonoid pathway. One of the most studied transcription factor complexes involved in this regulation consists of a MYB, bHLH and WD40. Chinensis), a popular monocot bulb flower, the regulatory mechanism of flavonoid biosynthesis remains unclear. Expression of genes encoding enzymes of flavonoid-specific biosynthesis is regulated by a conserved transcription factor complex, The bHLH transcription factor family is the second largest transcription factor family found in plants, which are distinguished by containing two functional regions in the protein. 200 amino acids at the N-terminus of the protein sequence is involved in interaction with the MYB transcription factor. The 200 amino acids bind to a WD40 protein and may contact with RNA polymerase II to produce transcriptional activation [12].

Methods

Results

Discussion

Conclusion