Molecular evolution analysis of MYB5 in Brassicaceae with specific focus on seed coat color of Brassica napus

Abstract

Background: MYB transcription factors are splay a vital role in plant biology, with previous research highlighting the significant impact of the R2R3-MYB-like transcription factor MYB5 on seed mucilage biosynthesis, trichome branching, and seed coat development. However, there is a dearth of studies investigating its role in the regulation of proanthocyanidin (PA) biosynthesis.

Results: In this study, a total of 51 MYB5 homologous genes were identified across 31 species belonging to the Brassicaceae family, with particular emphasis on Brassica napus for subsequent investigation. Through phylogenetic analysis, these genes were categorized into four distinct subclasses. Protein sequence similarity and identity analysis demonstrated a high degree of conservation of MYB5 among species within the Brassicaceae family. Additionally, the examination of selection pressure revealed that MYB5 predominantly underwent purifying selection during its evolutionary history, as indicated by the Ka/Ks values of all MYB5 homologous gene pairs being less than one. Notably, we observed a higher rate of non-synonymous mutations in orthologous genes compared to paralogous genes, and the Ka/Ks value displayed a stronger correlation with Ka. In B. napus, an examination of expression patterns in five tissues revealed that MYB5 exhibited particularly high expression in the black seed coat. The findings from the WGCNA demonstrated a robust correlation between MYB5 and BAN(ANR) associated with PA biosynthesis in the black seed coat, providing further evidence of their close association and co-expression. Furthermore, the results obtained from of the analysis of protein interaction networks offer supplementary support for the proposition that MYB5 possesses the capability to interact with transcriptional regulatory proteins, specifically TT8 and TT2, alongside catalytic enzymes implicated in the synthesis of PAs, thereby making a contribution to the biosynthesis of PAs. These findings imply a plausible and significant correlation between the nuique expression pattern of MYB5 and the pigmentation of rapeseed coats. Nevertheless, additional research endeavors are imperative to authenticate and substantiate these findings.

Conclusions: This study offers valuable insights into the genetic evolution of Brassicaceae plants, thereby serving as a significant reference for the genetic enhancement of Brassicaceae seed coat color.

Keywords: Brassica napus; Brassicaceae; Homologous gene; MYB5; Seed coat.

Fig. 1

Phylogenetic relationships of MYB5 proteins between 31 Brassicaceae species. All 4 subfamilies of MYB5 proteins were well separated in different clades and represented by different colors. IQ-TREE software (v1.6.12) to build a phylogenetic tree, where the -m parameter selects MFP, automatically detects the best model and builds a tree

Fig. 2

Phylogenetic, conserved motifs and promoter characteristics analysis of MYB5. Promoter sequences (-2,000 bp) of 51 MYB5 homologous genes were analyzed using PlantCARE. Different shapes and colors represent different elements, PAFM was used for conserved domain prediction, and phylogenetic, promoter characteristics, gene structure, conserved motifs have been drawn by TBtools

Fig. 3

Comparison of substitution rates between orthologous and paralogous MYB5 genes among 31 Brassicaceae species. A Comparative analysis of Ka, Ks and Ka/Ks of paralogous and orthologous MYB5 genes. B Correlation analysis between Ka, Ks and Ka/Ks of paralogous and orthologous MYB5 genes

Fig. 4

Expression analysis of four MYB5 homologous genes in Brassica napus five different tissues. The FPKM (Fragments Per Kilobase Million) value obtained from transcriptome sequencing data analysis was used to represent the expression pattern of the four copies of the MYB5 gene in different tissues, both five different tissues with three biological replicates

Fig. 5

The expression patterns of anthocyanin-related genes in five tissues of B. napus were analyzed using a heat map that represents the FPKM values of these genes. The colors on the heat map, ranging from red to pink and white, indicate the expression levels from high to low

Fig. 6

Co-expression analysis of genes related to anthocyanin biosynthesis was conducted in five tissues of B. napus. A Modular hierarchical clustering: The co-expression modules are depicted in different colors, while gray modules indicate no correlation between genes. B Module gene clustering heatmap: The gene expression network of anthocyanin biosynthesis-related genes in different tissues was analyzed using WGCNA, leading to the clustering of genes into distinct co-expression modules. C Module-to-sample correlation heatmap: Correlation analysis was performed between the co-expression modules of various genes associated with anthocyanin biosynthesis in different tissues. The numbers above the heat map indicate the Pearson correlation coefficient (r) values. D KEGG enrichment analysis of ‘turquoise’ module. E GO enrichment analysis of ‘turquoise’ module. F Cytoscape representation of co-expression network of the hub gene in the ‘turquoise’ module

Fig. 7

Protein-protein interaction network analysis of ‘turquoise’ module genes. Interactions between the proteins are represented by connecting lines, with the line thickness reflecting the strength of the interaction