Genome-Wide Identification and Characterization of bHLH Transcription Factors Related to Anthocyanin Biosynthesis in Red Walnut (Juglans regia L.).

Wei Zhao,Haijun Meng,Yonghui Liu,Lei Wang,Ying Yang,Zhaobin Dong,Guoliang Wu,Lin Li

doi:10.3389/fgene.2021.632509

Wei Zhao, Haijun Meng + Show 6 more

Open Access

https://doi.org/10.3389/fgene.2021.632509

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Abstract

Basic helix-loop-helix (bHLH) proteins are transcription factors (TFs) that have been shown to regulate anthocyanin biosynthesis in many plant species. However, the bHLH gene family in walnut (Juglans regia L.) has not yet been reported. In this study, 102 bHLH genes were identified in the walnut genome and were classified into 15 subfamilies according to sequence similarity and phylogenetic relationships. The gene structure, conserved domains, and chromosome location of the genes were analyzed by bioinformatic methods. Gene duplication analyses revealed that 42 JrbHLHs were involved in the expansion of the walnut bHLH gene family. We also characterized cis-regulatory elements of these genes and performed Gene Ontology enrichment analysis of gene functions, and examined protein-protein interactions. Four candidate genes (JrEGL1a, JrEGL1b, JrbHLHA1, and JrbHLHA2) were found to have high homology to genes encoding bHLH TFs involved in anthocyanin biosynthesis in other plants. RNA sequencing revealed tissue- and developmental stage-specific expression profiles and distinct expression patterns of JrbHLHs according to phenotype (red vs. green leaves) and developmental stage in red walnut hybrid progeny, which were confirmed by quantitative real-time PCR analysis. All four of the candidate JrbHLH proteins localized to the nucleus, consistent with a TF function. These results provide a basis for the functional characterization of bHLH genes and investigations on the molecular mechanisms of anthocyanin biosynthesis in red walnut.

Highlights

Walnut (Juglans regia L.) is among the top four economically and ecologically important nuts worldwide (Zheng et al, 2020)
We examined the subcellular localization of the four candidate anthocyanin biosynthesis-related transcription factors (TFs) (JrEGL1a, JrEGL1b, JrbHLHA1, and JrbHLHA2) in N. benthamiana transiently infiltrated with plasmids encoding the cDNA of the corresponding genes
The biosynthesis of anthocyanin depends on phenylalanine metabolism (Supplementary Figure S2) and involves the regulation of structural genes by the MBW complex formed by MYB, Basic helix-loop-helix (bHLH), and WD40 TFs (Baudry et al, 2004; Ma et al, 2018)

Summary

Introduction

Walnut (Juglans regia L.) is among the top four economically and ecologically important nuts worldwide (Zheng et al, 2020). The leaf and pericarp of the most widely cultivated walnut varieties are green and the seed coat is generally light yellow. The red walnut found in China has a red leaf, pericarp, seed coat, and xylem (Supplementary Figure S1; Li et al, 2018b). The molecular mechanisms of pigment formation in walnut have not yet been elucidated. Key enzymes encoded by structural genes have been shown to promote anthocyanin biosynthesis, including chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase, and anthocyanidin synthase; their spatial and temporal expression patterns are regulated by various transcription factors (TFs; Butelli et al, 2012). The functions of structural genes are for the most part conserved, and interspecies differences in the mechanisms of anthocyanin accumulation are attributable to the activities of distinct TFs

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