Genome-wide identification of MYBL2 in Brassicaceae, with a focus on the expression pattern of regulating anthocyanin synthesis in Brassica crops

Abstract

The Brassicaceae family includes the model plant Arabidopsis thaliana, along with various vegetables and oil crops, which possess significant economic and scientific value. Notably, three diploid species within the U's Triangle of Brassica have undergone natural hybridization, resulting in the formation of three allotetraploid species, which provides an excellent model for investigating the phylogenetic, evolutionary, and functional differentiation of plant homologous genes. In this study, we systematically identified MYBL2 homologous genes within the 31 Brassicaceae species, with a total of 48 homologous genes identified from 30 species, and phylogenetic analysis revealed the presence of six subfamilies, Ka/Ks analysis showed that only 10 MYBL2 homologous gene were positively selected during evolution. We subsequently concentrated on the evolution, gene structure, and conserved domain analysis of 16 MYBL2 homologous genes across six Brassica crops found in U's Triangle. Our findings indicated that these 16 MYBL2 homologous genes predominantly clustered into two branches and exhibited a high degree of evolutionary conservation. Further RNA-seq analysis of various tissues and organs from Brassica crops demonstrated that MYBL2 homologous genes were significantly up-regulated in tissues with anthocyanin accumulation. Concurrently, we employed Weighted Gene Co-expression Network Analysis (WGCNA) to identify hub genes regulated by anthocyanin in different tissues of B. napus, revealing that BnaMYBL2.C06 exhibited a strong repressor with anthocyanin biosynthetic genes (ABGs) in petals. Finally, quantitative reverse transcription PCR (qRT-PCR) analysis of B. napus leaves, stems, and petals indicated that four MYBL2 homologous genes were significantly up-regulated in leaves and petals, with expression patterns consistent with those of ABGs. Our results contribute new insights into the transcriptional regulatory mechanisms of anthocyanin in Brassica crops.

Keywords: Brassica crops; Brassicaceae; MYBL2; anthocyanins; expression pattern.

Figure 1

Phylogenetic relationships of MYBL2 homologous genes between 31 Brassicaceae species. The 48 homologous genes of MYBL2 were divided into six subfamilies, named Group I to Group VI by different colors, and AT1G66340.1 was divided into a separate group as an out group.

Figure 2

Evolution and collinearity analysis of 16 MYBL2 homologous genes in Brassica species. (A) Phylogenetic tree of 16 MYBL2 homologous genes; (B) Colinearity of 16 MYBL2 homologous genes among A, B and C subgenomes.

Figure 3

Analysis of cis-acting elements, gene structure, conserved motifs, conserved domains and protein sequence alignment of MYBL2 homologous gene promoters in six Brassica species. (A) Promoter cis-acting elements, gene structure, conserved motif and conserved domain analysis composite diagram, (a) promoter cis-acting elements, (b) gene structure, (c) conserved motif; (B) Conserved domain analysis of MYBL2 in six Brassica species, in the green box are R3 domain, EAR domain and TLLLF domain, respectively; (C) R3 conservative motif analysis MYBL2 in six Brassica species, in the red box are R3 motif, EAR motif and TLLLF motif.

Figure 4

Gene expression heat map of MYBL2 homologous genes in Brassica species. (A) Green and purple leaf of B. rapa; (B) Green and purple leaf of B. oleracea; (C) Green and purple leaf of B. juncea; (D) Different tissues of B. napus; (E) Different flower colors of B. napus; (F) Different developmental stages of B. napu purple petals.

Figure 5

Weighted gene co-expression network analysis of ABGs in different tissues of B. napus. (A) The cluster dendrogram tree showing three modules of co-expressed genes by WGCNA, each leaf of tree corresponds to one gene, and the tree branches constitute three modules, labeled with different colors; (B) The heat map results of module clustering and the relationship, a clustering algorithm is employed to group various co-expression modules, thereby illustrating the correlations among them, the correlation between modules is further depicted in the module correlation matrix below, both the horizontal and vertical axes represent gene modules, and a deeper red color indicates a higher correlation, while a greener color signifies a lower correlation between the modules; (C) Module-to-sample correlation heatmap, correlation analysis was performed between the co-expression modules of various genes associated with ABGs in different tissues, the numbers above the heat map indicate the Pearson correlation coeffcient (r) values; (D) Cytoscape representation of co-expressed anthocyanin metabolism related genes w in the MEturquoise module.

Figure 6

qRT-PCR analysis of expression patterns of anthocyanin biosynthesis genes in different tissues of B. napus. For qRT-PCR analysis, both white and purple flower samples are with three biological replicates, the point represents the mean value of three technical replicates in a representative biological experiment, the error bars indicate s.d, student’s t-test, **P<0.01, *P<0.05.