Genome-wide analysis of R2R3-MYB transcription factors in Boehmeria nivea (L.) gaudich revealed potential cadmium tolerance and anthocyanin biosynthesis genes

Abstract

Gene family, especially MYB as one of the largest transcription factor family in plants, the study of its subfunctional characteristics is a key step in the study of plant gene function. The sequencing of ramie genome provides a good opportunity to study the organization and evolutionary characters of the ramie MYB gene at the whole genome level. In this study, a total of 105 BnGR2R3-MYB genes were identified from ramie genome and subsequently grouped into 35 subfamilies according to phylogeny divergence and sequences similarity. Chromosomal localization, gene structure, synteny analysis, gene duplication, promoter analysis, molecular characteristics and subcellular localization were accomplished using several bioinformatics tools. Collinearity analysis showed that the segmental and tandem duplication events is the dominant form of the gene family expansion, and duplications prominent in distal telomeric regions. Highest syntenic relationship was obtained between BnGR2R3-MYB genes and that of Apocynum venetum (88). Furthermore, transcriptomic data and phylogenetic analysis revealed that BnGMYB60, BnGMYB79/80 and BnGMYB70 might inhibit the biosynthesis of anthocyanins, and UPLC-QTOF-MS data further supported the results. qPCR and phylogenetic analysis revealed that the six genes (BnGMYB9, BnGMYB10, BnGMYB12, BnGMYB28, BnGMYB41, and BnGMYB78) were cadmium stress responsive genes. Especially, the expression of BnGMYB10/12/41 in roots, stems and leaves all increased more than 10-fold after cadmium stress, and in addition they may interact with key genes regulating flavonoid biosynthesis. Thus, a potential link between cadmium stress response and flavonoid synthesis was identified through protein interaction network analysis. The study thus provided significant information into MYB regulatory genes in ramie and may serve as a foundation for genetic enhancement and increased productivity.

Keywords: MYB TFs; anthocyanin biosynthesis; cadmium stress; expression profiles; protein interaction; ramie.

FIGURE 1

Consensus sequence and the level of conservation of R2R3-MYB domains from ramie. The sequence logos of the R2 (A) and R3 (B) MYB repeats are based on full-length alignments of all ramie R2R3-MYB domains. The bit score exhibits the information content for each position in the sequence. The position of the three α-helices were marked (Helix 1–3). The conserved tryptophan residues (Trp, W) in the MYB domain are marked with black asterisks.

FIGURE 2

Phylogenetic relationships, gene structure and architecture of conserved protein motifs in BnGR2R3-MYB genes. (A) The maximum likelihood phylogenetic includes 105 R2R3-MYB proteins from ramie and 23 representatives from Arabidopsis (B) The motif architecture of ramie MYB proteins. 20 different motifs are displayed in different colored boxes. (C) Exon-intron structure of ramie MYB genes. Green boxes indicate exons; blue boxes indicate untranslated 5′- and 3′- regions; black lines indicate introns. R2, R3 MYB domain are highlighted by boxes of different shape and color.

FIGURE 3

Schematic representations for the chromosomal distribution and inter-chromosomal relationships of ramie R2R3-MYB genes. Gray lines indicate all synteny blocks in the ramie genome, and the red lines indicate duplicated MYB gene pairs. Blue lines indicate tandemly duplicated gene pairs. Gene and chromosome names are displayed on the tip and inside of the chromosome, respectively.

FIGURE 4

Synteny analyses of R2R3-MYB genes between ramie and five representative plants: Boehmeria nivea, Cannabis sativa, A. venetum, O. sativa, Zea mays and A. thaliana. Gray lines in the background indicate the collinear blocks within ramie and other plant genomes, while the red lines highlight the syntenic R2R3-MYB gene pairs.

FIGURE 5

Neighbor-joining (Guo et al., 2019) tree showing relationships among R2R3-MYB proteins from Boehmeria nivea (Chen et al., 2015b), A. thaliana (At), O. sativa (Os), Zea mays (Pierrugues et al., 2001), S. lycopersicum (Sl) and A. comosus (Ac). MYB proteins from the six species were designated as C1 to C35. The table on the right indicated the number of the subgroup members and function of clades in each species.

FIGURE 6

Analysis of cis-elements from promoter region of the BnGR2R3-MYB genes. The figure on the left shows the distribution of four cis-acting elements upstream promoter regions (1000 bp). On the right is the heat map of the quantity of plant growth, stress responsive and phytohormone responsive cis-elements, represented in brown, blue and green, respectively, circle sizes indicate the number of cis-elements.

FIGURE 7

Hierarchical clustering of expression profiles of ramie R2R3-MYB genes in 9 samples including different tissues and developmental stages. Log2(FPKM +1) values were displayed according to the color code. Detailed FPKM values were listed in Supplementary Table S7.

FIGURE 8

Phylogenetic tree and gene expression analysis of R2R3-MYB transcription factors associated with anthocyanin synthesis.

FIGURE 9

The relative expression levels of selected MYB genes in two ramie varieties in different tissue sites (root, stem, leaf) at different periods under Cd⁺² treatment (0, 6, 12, 24, 48 h). (A) represents root; (B) represents stem; (C) represents leaf. The error bar represents the standard deviation of the three biological duplicates. * represents significant difference (p < 0.05), ** represents extremely significant difference (p < 0.01).

FIGURE 10

The protein-protein interaction network for BnGR2R3-MYB proteins based on their Arabidopsis orthologs. The red solid circles indicated cadmium stress-related MYB transcription factors, the olive squares represented stress-response proteins, and the green squares were flavonoid synthesis related proteins.