Comparative genomics and transcriptomics analysis of the bHLH gene family indicate their roles in regulating flavonoid biosynthesis in Sophora flavescens

Abstract

The basic helix-loop-helix (bHLH) transcription factors play crucial roles in various processes, such as plant development, secondary metabolism, and response to biotic/abiotic stresses. Sophora flavescens is a widely used traditional herbal medicine in clinical practice, known for its abundant flavonoids as the main active compounds. However, there has been no comprehensive analysis of S. flavescens bHLH (SfbHLH) gene family reported currently. In this study, we identified 167 SfbHLH genes and classified them into 23 subfamilies based on comparative genomics and phylogenetic analysis. Furthermore, widespread duplications significantly contributed to the expansion of SfbHLH family. Notably, SfbHLH042 was found to occupy a central position in the bHLH protein-protein interaction network. Transcriptome analysis of four tissues (leaf, stem, root and flower) revealed that most SfbHLH genes exhibited high expression levels exclusively in specific tissues of S. flavescens. The integrated analysis of transcriptomics and metabolomics during pod development stages revealed that SfbHLH042 may play a central role in connecting SfbHLH genes, flavonoids, and key enzymes involved in the biosynthesis pathway. Moreover, we also checked the expression of 8 SfbHLH genes using RT-qPCR analysis to realize the expression profiles of these genes among various tissues at different cultivated periods and root development. Our study would aid to understand the phylogeny and expression profile of SfbHLH family genes, and provide a promising candidate gene, SfbHLH042, for regulating the biosynthesis of flavonoids in S. flavescens.

Keywords: Sophora flavescens; bHLH; coexpression network; flavonoid biosynthesis; gene duplication.

Figure 1

Conserved motif analysis of bHLH domain in S. flavescens. The overall height of each stack represents the conservation degree of the sequences at the position. The AA site with a conservation ratio more than 50% was identified with capital letters.

Figure 2

Evolutionary analysis of bHLH genes among five species. (A) Phylogenetic tree of bHLH proteins in S. flavescens, A. thaliana and rice; (B). Phylogenetic tree of bHLH proteins in the S. flavescens, S. moorcroftiana and S. japonica; (C). The number comparison of 23 subfamilies among the S. flavescens, S. moorcroftiana and S. japonica; (D). Gene expansion and contraction among five species (CAFE 5); (E). The standard deviations (SD) of bHLH genes per subfamily among five species are positively correlated with the average size of each subfamily.

Figure 3

Gene duplication pattern and syntenic analyses of bHLH genes in S. flavescens, S. moorcroftiana and S. japonica. (A). The locations of each SfbHLH gene on 9 chromosomes and 1 scaffold. To avoid crowding, we only show the names of these SfbHLH genes experienced WGD, TRD and DSD events on the Circos diagram. The different colors (red to yellow) in the middle track indicated high to low gene density. (B). The collinear relationship between S. flavescens, S. moorcroftiana, and S. japonica. The collinear blocks are represented by gray lines, while the bHLH gene collinearity is indicated by green and red lines. S. fla, S. moo, and S. jap were short for S. flavescens, S. moorcroftiana, and S. japonica, respectively.

Figure 4

Protein-protein network and function analysis of SfbHLHs. (A) Protein-protein network for SfbHLHs constructed according to information available about Arabidopsis bHLHs in STRING database through orthologous relationship. (B). KEGG enrichment analysis of SfbHLH genes. The size of the dot indicates the bHLH number in certain pathway, and the color reflects the p value.

Figure 5

Expression patterns of SfbHLH genes among different tissues. (A) The expression profile of SfbHLHs among four tissues, including root, stem, leaf and flower. (B). K-means cluster analysis of the expression of SfbHLHs among four tissues. Those SfbHLHs with expression values less than 1 in all the four tissues were excluded from the cluster analysis.

Figure 6

Expression profiles of the S. flavescens pods at different developmental periods. (A). The expression profile of SfbHLHs during the pod development. (B). K-means cluster analysis of the expression of SfbHLHs and key enzymes in the flavonoid synthesis pathway, and the accumulation of the S. flavescens flavonoids during the pod development. (C). The co-expression network of SfbHLHs, key enzymes and flavonoids. The red dot represents the SfbHLH gene, the green dot represents flavonoid metabolite, and the yellow dot represents key enzyme in the flavonoid synthesis pathway. (D). The expression of hub SfbHLH genes during the pod development. (E). The co-expression network of SfbHLH091, key enzymes and flavonoids. (F). The KEGG enrichment analysis of genes co-expressed with SfbHLH091.

Figure 7

Expression analysis of SfbHLH genes during the root development and among different tissues with different cultivated years by RT-qPCR. (A–H). The expression levels of different SfbHLH genes during the root development. (I–P). The expression levels of different SfbHLH genes among different tissues with different cultivated years. Each sample contain three biological replicates. Different letters on different columns indicate a significant difference between any two samples (p < 0.05).