Genome-Wide Identification and Expression Analysis of the R2R3-MYB Transcription Factor Family Revealed Their Potential Roles in the Flowering Process in Longan (Dimocarpus longan)

Abstract

Longan (Dimocarpus longan Lour.) is a productive fruit crop with high nutritional and medical value in tropical and subtropical regions. The MYB gene family is one of the most widespread plant transcription factor (TF) families participating in the flowering regulation. However, little is known about the MYB TFs involved in the flowering process in longan and its regulatory network. In this study, a total of 119 DlR2R3-MYB genes were identified in the longan genome and were phylogenetically grouped into 28 subgroups. The groupings were supported by highly conserved gene structures and motif composition of DlR2R3-MYB genes in each subgroup. Collinearity analysis demonstrated that segmental replications played a more crucial role in the expansion of the DlR2R3-MYB gene family compared to tandem duplications, and all tandem/segmental duplication gene pairs have evolved under purifying selection. Interspecies synteny analysis among longan and five representative species implied the occurrence of gene duplication events was one of the reasons contributing to functional differentiation among species. RNA-seq data from various tissues showed DlR2R3-MYB genes displayed tissue-preferential expression patterns. The pathway of flower development was enriched with six DlR2R3-MYB genes. Cis-acting element prediction revealed the putative functions of DlR2R3-MYB genes were related to the plant development, phytohormones, and environmental stresses. Notably, the orthologous counterparts between Arabidopsis and longan R2R3-MYB members tended to play conserved roles in the flowering regulation and stress responses. Transcriptome profiling on off-season flower induction (FI) by KClO₃ indicated two up-regulated and four down-regulated DlR2R3-MYB genes involved in the response to KClO₃ treatment compared with control groups. Additionally, qRT-PCR confirmed certain genes exhibited high expression in flowers/flower buds. Subcellular localization experiments revealed that three predicted flowering-associated MYB proteins were localized in the nucleus. Future functional studies on these potential candidate genes involved in the flowering development could further the understanding of the flowering regulation mechanism.

Keywords: Dimocarpus longan; KClO3; R2R3-MYB; flower development; qRT-PCR.

FIGURE 1

Characterization of R2 (A) and R3 (B) MYB repeats of longan R2R3-MYB proteins. The position score indicated the degree of conservation of each amino acid at the specific position within sequences. The positions of the three α-helices are marked (helices 1–3). Arrows represent amino acid insertion sites detected in the R2 repeat of genes DlMYB23, DlMYB27, DlMYB75, and DlMYB98 and the R3 repeat of DlMYB53, DlMYB54, and DlMYB93. The length of each insertion (amino acid, aa) is highlighted in parentheses. Characteristic tryptophan (W) residues are marked with the symbol “*”.

FIGURE 2

Phylogenetic analysis of longan and Arabidopsis MYB proteins. The full-length amino acid sequences of longan (119) and Arabidopsis (126) MYB proteins were compared using RAxML software to generate ML phylogenetic trees with 1,000 bootstrap values. The Evolview3 was used to visualize and display > 50 bootstrap values at the nodes. Red and blue stars represent DlMYB and AtMYB proteins, respectively. All MYB proteins were divided into 28 subgroups (S1–S28).

FIGURE 3

Phylogenetic tree, conserved motifs, gene structural, and cis-acting element analysis of DlMYBs. (A) An Maximum Likelihood (ML) tree of 119 DlMYBs proteins with 1,000 bootstrap replicates. (B) The conserved motifs in DlMYBs were predicted using the MEME Suite web server. (C) Exon/intron structures of the longan R2R3-MYB genes. The introns, exons, and UTRs are indicated by orange boxes, black lines, and blue boxes, respectively. (D) The cis-acting elements of the longan R2R3MYB proteins were predicted using PlantCARE.

FIGURE 4

Chromosomal location, tandem duplication, and segmental gene duplication analysis of the DlMYB genes. (A) The distribution map of DlMYB genes on the 15 chromosomes of longan (genes with tandem clusters are marked in red). (B) Circos plot of DlMYB genes in the longan genome. Gene pairs with segmental duplications (SDs) are indicated by blue lines, and gene density was highlighted by red rectangular boxes.

FIGURE 5

Collinearity analysis of the DlMYB genes. (A) Synteny relationship of R2R3MYB genes between longan and five other representative plant species: Arabidopsis, litchi, yellow horn, maize, and rice. Gray lines in the background indicate the collinear blocks within two genomes, and red lines highlight the syntenic MYB gene pairs. (B) The R2R3MYB genes formed the colinear pairs between longan and the other five species. Bar graphs indicate the number of genes in colinear pairs.

FIGURE 6

Nuclear localization of DlMYB16, DlMYB72, and DlMYB116 proteins in Arabidopsis protoplasts. GFP: green fluorescent protein; mKATE: nuclear marker; Cy5: chloroplast; Bright: bright field; merged: combined mKATE and GFP and bright field. Bars, 20 μm.

FIGURE 7

Tissue-specific expression and GO enrichment analysis of DlMYB genes. (A) The expression levels of DlMYB genes in nine tissues were classified into groups I–VII. Different colors indicate log2 transformed expression levels, with red indicating high expression levels and blue indicating low expression levels. (B) Some DlMYB genes were classified as related to stress response, development, and hormones. The size of the circle indicates the number of DlMYB genes, and the color gradient indicates the size of the P-value. Six DlMYB genes are categorized into “flower development” function. A red pentagram represents that the gene is relevant, while a black pentagram indicates that the gene is not involved in the category.

FIGURE 8

Expression patterns of DlMYB genes under KClO₃ treatment. (A) The heat map displays the hierarchical clustering of the 119 DlMYB genes at different time points, with red indicating high expression levels and blue indicating low expression. (B) The line graphs display the effect of KClO₃ treatment on the expression of DlMYB genes. The blue line represents the control and the pink line represents KClO₃ treatment. (C) The heatmap from WGCNA of differentially expressed genes (DEGs) in off-season flower induction at different treatments and developmental stages. Each row corresponds to one module resulting from an WGCNA eigengene value. Each column corresponds to the control treatment and the KClO₃ treatment at different time-points. The color and number of each cell at the row-column intersection indicate the correlation coefficient between one specific module and one time point under control or KClO₃ treatment. Darker red represents a higher degree of correlation. The number in brackets indicate the significance (p-value) of the correlation coefficient. The number of DlMYB genes in each module is shown in the leftmost column.

FIGURE 9

qRT-PCR and RNA-seq analyses of eight selected DlMYB genes. The samples from flowers, flower buds, leaves, pulps, and pericarps show a range of expression changes of selected DlMYB genes. The left and right sides of the Y-axis indicate relative expression levels and TMM, respectively. To confirm the tissue-specific expression of the DlMYB genes, the expression pattern of selected DlMYB genes was calculated as a fold relative to the expression level of the pericarp.