Systematic analysis of potato MYB family genes and their potential functions in anthocyanin biosynthesis

Abstract

MYB proteins, one of the largest transcription factor superfamilies in plant kingdom, play essential roles in plant development and metabolic regulation, including anthocyanin biosynthesis. MYB family genes have been widely identified in many plant species, but which have not yet been comprehensively identified and systematically analyzed in the latest potato genome (DM v6.1, the PGSC 2020). In this study, a total of 294 StMYB genes were identified, including 149 St1R-MYBs, 142 St2R-MYBs, and three St3R-MYBs. The 142 St2R-MYB proteins were classified into 20 subgroups, and similar protein motifs were found within the same subgroup. A total of 12 candidate St2R-MYB genes were screened out by phylogenetic clustering analysis for regulating anthocyanin biosynthesis. Among them, four genes StMYB_66, StMYB_99, StMYB_150 and StMYB_248 had negative regulatory functions, while the functions of four genes StMYB_240, StMYB_241, StMYB_242 and StMYB_243 were opposite in anthocyanin biosynthesis through tobacco transient transformation. These results laid the foundation for further study on biological functions and molecular mechanisms of the St2R-MYB genes regulating anthocyanin biosynthesis in potato, and provided theoretical basis and new gene resources for the breeding of colorful potato varieties.

Keywords: StMYB; Anthocyanin biosynthesis; Functional analysis; Potato genome.

Fig. 1

Distribution of 294 StMYB genes on chromosomes of DM potato. The chromosome name is given to the left of each chromosome. Gray oval on the chromosome bar represents centromere location for each chromosome, information of centromeres was collected according to the PGSC 2020. The tandem duplication pair of two StMYB genes was marked by red line.

Fig. 2

The subcellular localization of StMYB_99, StMYB_150, and StMYB_243 in tobacco epidermal cells. The panels from left to right correspond to fluorescence (GFP), bright field (Bright) and merged fluorescence (Merged) images, respectively. Scale bars: 20 µm (red) and 50 µm (white).

Fig. 3

Interchromosomal relationships of StMYB genes from DM potato. The 72 putative segmental duplication pairs of the StMYB genes were linked by the red lines.

Fig. 4

Synteny analyses of MYB genes between DM potato and six representative plant species. The synteny analysis was performed and visualized using the MCScanX and TBtools software, respectively. Gray lines in the background represented collinear blocks within DM potato and other representative plant species, while colored lines exhibited syntenic MYB gene pairs. Chromosomes of six representative plant species were showed by different colored rectangles.

Fig. 5

Analyses of phylogenetic relationships, gene structures, conserved motifs, and cis-acting elements of St2R-MYB genes. (A) Phylogenetic tree of the 142 St2R-MYB proteins. (B) Exon–intron structures of the St2R-MYB genes. Yellow boxes indicated 5′-UTR and 3′-UTR regions, and red boxes and gray lines represented exons and introns, respectively. (C) Motif compositions of the St2R-MYB proteins. Twenty different types of motifs were exhibited in different colored boxes. The scale at the bottom can be used for estimation of the protein lengths. (D) Distribution of cis-acting elements of St2R-MYB genes promoters. Twenty-eight different types of responsiveness elements were displayed in different colored blocks.

Fig. 6

Unrooted phylogenetic treebetween St2R-MYB genes and 2R-MYB genes from other species. The 2R-MYB genes highlighted in orange, pink, red, dark blue, dark green, yellow, blue, light green, and light yellow were from DM potato, tomato, eggplant, pepper, tobacco, grape, Arabidopsis, and petunia, respectively. “ + ” represents anthocyanin related promoter genes, while “-” represents anthocyanin related inhibitor genes. The phylogenetic tree was constructed using MEGA 11 by the neighbor-joining (NJ) algorithm method with 1,000 replicates of bootstrap value, and visualized using the ChiPlot software.

Fig.7

Transient assays of the candidate St2R-MYB genes. Phenotype and anthocyanin content in tobacco leaves collected 7 days after infiltration with Agrobacterium harboring StMYB_240, StMYB_241, StMYB_242, StMYB_243, StMYB_66, StMYB_99, StMYB_150, and StMYB_248, an empty vector (EV) as a control. The error bars means the standard deviation of three biological replicates. Statistical significance was analyzed by the Student’s t-test: ** P < 0.01 (A–D) or by one-way ANOVA followed by the Duncan’s post-hoc test using SPSS software, different lowercase letters indicate significant differences (P < 0.01) (E–H). Degrees of freedom and F-values were showed in Table S10.